Systems and methods for recording simultaneously visible light image and infrared light image from fluorophores

ABSTRACT

The invention provides systems and methods for imaging a sample. In various embodiments, the invention provides a system comprising an image sensor, a laser for emitting excitation light for an infrared or near-infrared fluorophore, a visible light source, a notch beam splitter, a notch filter, a synchronization module, an image processing unit, an image displaying unit, and light-conducting channels. In various embodiments, the present invention provides a system comprising an image sensor, a laser for emitting excitation light for an infrared or near-infrared fluorophore, a laser clean-up filter, a notch filter, a white light source, an image processing unit, an image displaying unit, and light-conducting channels. In accordance with the present invention, the image sensor can detect both visible light and infrared light.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International PatentApplication No. PCT/US2014/035203, filed Apr. 23, 2014, which designatedthe U.S., was published under PCT Article 21(2) in English, and claimspriority under 35 U.S.C. §119(e) to U.S. provisional patent applicationNo. 61/814,955, filed Apr. 23, 2013. This application also claimspriority under 35 U.S.C. §119(e) to U.S. provisional patent applicationNo. 62/049,312, filed Sep. 11, 2014. The contents of all the relatedapplications cross-referenced herein are herein incorporated byreference in their entirety as though fully set forth.

FIELD OF INVENTION

The invention provides systems and methods for recording simultaneouslyvisible light image and infrared (IR) light image from fluorophores.

BACKGROUND OF THE INVENTION

All publications cited herein are incorporated by reference in theirentirety to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. The following description includesinformation that may be useful in understanding the present invention.It is not an admission that any of the information provided herein isprior art or relevant to the presently claimed invention, or that anypublication specifically or implicitly referenced is prior art.

In recent years, there has been an interest in the use of infrared (IR)dyes for detection of tagged tissue such as tumors and vessels duringsurgical removal of tumors in a clinical setting. Infrared dyes areconsidered superior tagging dyes for marking tissue due to their higherpenetration depths, lack of auto-fluorescence in that region of spectrumthat can add noise to the imaging, and also lack of absorption fromhemoglobin (i.e., blood) and water in that region of the spectrum whichcan reduce the fluorescence signal. To utilize these dyes in, forexample, the clinical operating room environment requires an IRsensitive imaging system, which is capable of acquiring high resolutionimages in the normal white light visible spectrum, while simultaneouslyacquiring and overlaying the infrared signal on top of normal visiblespectrum images in order to provide a contrast to a surgeon whileoperating.

However, due to the general absence of applications of fluorescent tumorligands in surgical oncology, currently there are no imaging systemsavailable commercially that are optimized for near infrared (NIR)fluorescence based resection of tumors. The clinical systems that doexist were primarily designed to detect unbound intravascularindocyanine green (ICG), an FDA approved NIR fluorescent dye. ICG istypically intravenously administered in high doses, and imaging isperformed 30-60 minutes after injection. The intravascular fluorescentload achieved with this approach is high, and approved clinical imagingdevices have adequate sensitivity for these applications. Examples ofsuch systems include a fluorescent module incorporated into operatingmicroscopes (OPMI Pentero Infrared 800, Carl Zeiss) as well at the SPY®and Pinpoint® systems (Novadaq), and the FluoBeam® 800 (Fluoptics)hand-held unit.

These systems have adequate sensitivity for intravascular imaging, butare not practical for use in, for example, targeted tumor-specific NIRfluorescence. For example, Fluobeam is hand held device with no overlayof white light images but is not designed for practical use as asurgical tool that requires HD quality images in white light,maneuverability, magnification, illumination, and automatedco-registration of NIR images. One of the reasons for such lowsensitivity is due to less fluorescent photons captured by the imagingsystem, as such systems may principally use one (NIR only) or two (NIRand visible) cameras with a long pass filter. In a simultaneous visibleand NIR capture imaging systems, one camera captures the image in thevisible spectrum and second camera captures the fluorescent image. Thisis achieved by splitting the incident light from the field into twochannels using a beam-splitter. One beam transmits the NIR fluorescentlight to one of the cameras while the other beam of visible light passesthrough the beam splitter into the second camera. As the fluorescentexcitation and emission of NIR dyes such as ICG have a very narrowstokes shift, the long pass filter causes a significant loss offluorescent light (FIG. 1), and subsequent detection sensitivity.Fluorescence imaging of tumors requires a targeting moiety to attainhigh specificity, and enable reliable differentiation between cancertissue and surrounding normal tissues. To achieve this, doses are keptlow and the time between drug administration and imaging is quite long(12-48 hours in most cases) to permit uptake of the probe by the tumorand for the washout of unbound material from normal tissues. Thisresults in markedly less fluorescent signal, making currently marketedsystems inadequate for detection. Additionally, these systems can becumbersome to use in the clinical setting, due to the fact that thereare two camera attachments, and require a complete change in theexisting setup. This inadequacy of the existing systems drives the needfor device innovation to take advantage of the specificity of thesenovel imaging agents.

Accordingly, there is a need for highly sensitive systems and methodsthat can record simultaneously visible light image and infrared lightimage from fluorescent dye. The invention described herein meets theunmet need by providing systems and methods for recording simultaneouslyvisible light image and infrared light image from fluorophores.

SUMMARY OF THE INVENTION

Various embodiments of the present invention provide an imaging systemfor imaging a sample comprising an infrared or near-infrared fluorophoreeither alone or attached to a targeting moiety such as a peptide,protein, nanoparticle, nanoconjugate, antibody, and nucleic acid (e.g.,DNA and RNA strands) or to any other such biologically specifictargeting entity. The imaging system comprises: an image sensor, alaser, a laser clean-up filter, a notch filter, and a white lightsource. The image sensor detects visible light and infrared light andgenerates sensor signals. The laser emits an excitation light for theinfrared fluorophore. The laser clean-up filter is placed in the lightpath from the laser to the sample, and narrows the wavelength band ofthe excitation light to the peak absorption band of the infrared ornear-infrared fluorophore. The narrowed excitation light excites theinfrared or near-infrared fluorophore at the peak absorption in thesample to emit an emission light. The notch filter is placed in thelight path from the sample to the image sensor, and blocks theexcitation light. The white light source emits a light comprisingvisible light. In various embodiments, the image sensor is without a NIRlong pass filter. In various embodiments, the imaging system furthercomprises a fast trigger unit.

Various embodiments of the present invention provide an imaging systemfor imaging a sample comprising an infrared or near-infraredfluorophore. The system comprises: an image sensor, a laser, a notchbeam splitter, a notch filter, and a synchronization module. The imagesensor detects visible light and infrared light and generates sensorsignals. The laser emits an excitation light for the infrared ornear-infrared fluorophore and alternates between on and off statuses.The notch beam splitter is placed in the light path from the laser tothe sample and in the light path from the sample to the image sensor.The excitation light is reflected by the notch beam splitter to thesample; the excitation light excites the infrared or near-infraredfluorophore in the sample to emit an emission light; and the emissionlight is transmitted through the notch beam splitter to the imagesensor. The notch filter is placed in the light path from the sample tothe image sensor, and the notch filter blocks the excitation light. Thesynchronization (trigger) module synchronizes the image sensor with thelaser and visible light, whereby a single sensor signal is synchronizedto a single on or off status of the laser.

Also provided is a method of imaging a sample. The method comprises thesteps of: providing a sample, providing an imaging system describedherein, and imaging the sample with said imaging system.

While various embodiments of the present invention are described in thecontext of various infrared or near-infrared fluorophores, it should notbe construed that the present invention is limited to those infrared ornear-infrared fluorophores. In fact, those infrared or near-infraredfluorophores are merely non-limiting examples. Indeed, the presentinvention may be used for fluorophores in any suitable segment ofelectromagnetic spectrum, for example, ultraviolet (UV), ultraviolet A,ultraviolet B, ultraviolet C, near ultraviolet, middle ultraviolet, farultraviolet, hydrogen lyman-alpha, vacuum ultraviolet, extremeultraviolet, visible, infrared, near infrared, mid infrared, and farinfrared. Examples of fluorophores outside the infrared or near-infraredrange include but are not limited to fluorescein, sodium yellow, and5-aminolevulinic acid (5-ALA). While in various embodiments of thepresent invention, particular types of imaging components (e.g., imagesensors, lasers, laser clean-up filters, notch filters, and otherassociated filters) are described in the context of various infrared ornear-infrared fluorophores, it should not be construed that the presentinvention is limited to those particular imaging components. In fact,those particular imaging components are merely non-limiting examples.Indeed, the present invention also contemplates choosing and includingappropriate imaging components (e.g., image sensors, lasers, laserclean-up filters, notch filters, and other associated filters) for theuse of those fluorophores outside the infrared or near-infrared range.

While various embodiments of the present invention are described in thecontext of imaging, diagnosing, and/or treating tumors, it should not beconstrued that the present invention is limited to such applications. Infact, the present invention may find utility in any and all detectionand diagnosis of a tissue difference, i.e., normal vs. abnormal, due toany and all reasons including but not limited to tumor, injury, trauma,ischemia, infection, inflammation, or auto-inflammation. The presentinvention provides imaging systems and systems for a wide range ofapplications, including but not limited to, imaging, diagnosing and/ortreating tumor tissues, injured tissues, ischemic tissues, infectedtissue, and inflammatory tissues. In any situation where a tissue ofinterest (e.g., a cancerous, injured, ischemic, infected, orinflammatory tissue) is different from the surrounding tissue (e.g.,healthy tissues) due to physiological or pathological causes, aninfrared or near-infrared fluorophore may be used to differentiallylabel the tissue of interest and the surrounding tissue, and those areasmay be imaged with the imaging systems and methods of the presentinvention to provide visual guidance for appropriate diagnosis andtreatment. Therefore, the imaging systems and methods may be used toimage, diagnose, and/or treat subjects with various conditions includingbut not limited to tumors, cancers, traumatic brain injury, spinal cordinjury, stroke, cerebral hemorrhage, brain ischemia, ischemic heartdiseases, ischemic reperfusion injury, cardiovascular diseases, heartvalve stenosis, infectious diseases, microbial infections, viralinfection, bacterial infection, fungal infection, and autoimmunediseases. The imaging systems of the present invention may also be usedto image normal tissues in a healthy subject, for example, to identifyvasculatures.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 depicts, in accordance with various embodiments of the presentinvention, the possible loss of fluorescent light when using of longpass filter for a two camera solution.

FIG. 2 depicts, in accordance with various embodiments of the presentinvention, the typical sensitivity of the color sensors.

FIG. 3 depicts, in accordance with various embodiments of the presentinvention, the color filter array over the image sensor.

FIG. 4 depicts, in accordance with various embodiments of the presentinvention, an exemplar system for simultaneously recording visible lightimage and infrared light image from fluorescent dye. The systemcomprises a laser 01 with a wavelength of 785 nm, a notch beam splitter@ 785 nm 02, a notch filter @ 785 nm 03, a CCD camera without IR filter04, and trigger or synchronization unit 05. The laser can alternatebetween the on and off statues at a frequencies about half the speed ofa CCD camera (for example 60 Hz). The CCD camera captures image framesat a frequency of 120 Hz. The synchronization unit synchronizes the CCDimage sensor with the laser to ensure that a single image framecorresponds to a single on or off status of the laser. The tissue istagged with an IR (or NIR) fluorophore. A visible light source 06illuminates the sample of interest. The wavelength of 785 nm is anon-limiting example, and other wavelengths can also be used with thissystem.

FIGS. 5A-5C depict, in accordance with various embodiments of thepresent invention, an exemplar method for simultaneously recordingvisible light image and infrared light image from fluorescent dye. Whenthe laser is off, the charge coupled device (CCD) camera captures Frame1, in which Red-Green Blue (RGB) pixel sensors detect visible light butno fluorescence in near infrared range (NIR). When the laser is on, theCCD camera captures Frame 2, in which RGB pixel sensors detect bothvisible light and additional fluorescence in NIR. The difference ofsubtracting Frame 1 from Frame 2 represents the additional fluorescencein NIR. This calculated frame of the additional fluorescence can begiven a false color and added back to Frame 1, thereby generating acomposite image frame of visible light and infrared light to bedisplayed to a surgeon. The process can be continuously repeated to showand record a real-time video during surgery.

FIGS. 6A-6C depict, in accordance with various embodiments of thepresent invention, a non-limiting example of clinical prototype. (FIG.6A) Design and optical specifications. A laser 01 emits an excitationlight for an infrared or near-infrared fluorophore. The excitation lighttravels into the camera and is reflected by a fold mirror 08 to a laserclean-up filter 07. Through the laser clean-up filter 07, the excitationlight is narrowed to the excitation wavelength of the infrared ornear-infrared fluorophore. The narrowed excitation light is reflected bya notch beam splitter 02, is reflected by another fold mirror 08, passesthrough a variety of optical components (for example, a collimating lens09 and a diffuser 10), and exits a window 11 of the camera toward asample. The narrowed excitation light excites the infrared ornear-infrared fluorophore in the sample to emit an emission light. Theemission light travels into the camera through another window 11, isreflected by a folder mirror 08 to a notch filter 03, and passes thenotch filter 03 and a variety of optical components (for example, aVIS-NIR lens 12). Through the notch filter 03, any excitation lightreflected from the sample is blocked. The emission light reaches animage sensor (for example, a camera manufactured by Basler Inc., or anyother suitable camera) that detects the excitation light and generates asensor signal. The emission light generated sensor signal is transferredfrom the camera via a data link to an image processing unit forgenerating an infrared image frame. A white light source 06 emits avisible light. The visible light travels into the camera, passes a notchbeam splitter 02, is reflected by a fold mirror 08, passes through avariety of optical components (for example, a collimating lens 09 and adiffuser 10), and exits a window 11 of the camera toward the sample. Thesample is illuminated by the visible light. The visible light travelsback into the camera through another window 11, is reflected by anotherfolder mirror 08 to a notch filter 03, and passes the notch filter 03and a variety of optical components (for example, a VIS-NIR lens 12).The visible light reaches an image sensor (for example, a cameramanufactured by Basler Inc., or any other suitable camera) that detectsthe visible light and generates a sensor signal. The visible lightgenerated sensor signal is transferred from the camera to an imageprocessing unit for generating a visible image frame. (FIG. 6B) Field ofillumination for the custom integrated lens and camera solution. In onenon-limiting example, the unit may measure 7.75″×3.74″×2.06″ and mayweight approximately 3.8 lbs allowing it to be attached to commercialendoscope holders. In one non-limiting example, with a focal distance ofabout 45 cm, it may sit far outside the surgical field and allowinstruments and specimen to be easily passed under it during surgicalexcision. The camera output is connected to an image processing computerand then fed to HD video monitor for display. (FIG. 6C) A scheme of theimaging system. An excitation light for an infrared or near-infraredfluorophore is emitted from a laser, and through the firstlight-conducting channel, is cleaned up by a laser clean-up filter andreaches a sample labeled with the infrared or near-infrared fluorophoreto excite the infrared or near-infrared fluorophore. An emission lightis emitted from the excited infrared or near-infrared fluorophore in thesample, and through the third light-conducting channel, passes through anotch filter and reaches an image sensor. A visible light is emittedfrom a white light source, and through the second light-conductingchannel, reaches and illuminates the sample. The visible from theilluminated sample, through the fourth light-conducting channel, reachesthe image sensor. The first, second, third and fourth channels mayinclude various optical components including but not limited to opticalfibers, optical filters, optical enhancers, optical attenuators, beamsplitters, condensers, diffusers, windows, holes, mirrors, shutters, andlens. They may overlap partially or completely; they may be separatechannels or combined into one, two, or three channels; and they mayinclude a device such as endoscope and microscope or a portion of thedevice. The image sensor detects the emission light to generate aninfrared light-based sensor signal and detects the visible light togenerate a visible light-based sensor signal. The image sensor isconnected to an image processing unit and transfers the sensor signalsto the image processing unit. The image processing unit processes thesensor signals to generate a composite image frame of infrared light andvisible light and transfers the composite image frame to an imagedisplaying unit, which displays a composite image of infrared light andvisible light. The imaging system continuously provides a stream ofcomposite images as a real-time video, for example, to assist a surgeonwith removing a tumor.

FIG. 7 depicts, in accordance with various embodiments of the presentinvention, a non-limiting example of filter configuration. The use ofvery narrow band laser light to excite ICG at the peak absorptionwavelength of 785 nm aided by use of a clean-up filter allows formaximum excitation efficiency. In conjunction a notch filter in front ofthe camera is able to remove the excitation light from the image thuscapturing only the fluorescence emission from the target. Thisconfiguration allows for imaging fluorescence with maximum efficiencywith high SNR.

FIG. 8 depicts, in accordance with various embodiments of the presentinvention, a non-limiting example of timing details of frame capture.This figure shows the timing details of 10 captured frames which areprocessed to produce a single displayed frame. The camera capturesframes at 300 frames per second, while the video display displays 30frames per second. Each captured frame is synchronized with the whitelight and NIR laser turning “ON” and “OFF”. The visible or natural lightframe is captured when the laser is “off” (no fluorescence) and onlywhite light is “ON”. When both light sources are “OFF” then SIRIScaptures the stray light (background). This background is subtractedfrom the fluorescence frame when only the laser in “ON” and the whitelight is “OFF”. Dividing this frame capture into groups of 5 frames eachreduces the ghosting effect during camera movement.

FIG. 9 depicts, in accordance with various embodiments of the presentinvention, a non-limiting example of a device or a computer systemcomprising one or more processors and a memory storing one or moreprograms for execution by the one or more processors.

FIG. 10 depicts, in accordance with various embodiments of the presentinvention, an imaging system for visualizing tumors. In this example,the image sensor is mounted at the patient end of an endoscope.

DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Allen et al., Remington: The Science and Practice of Pharmacy22^(nd) ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al.,Introduction to Nanoscience and Nanotechnology, CRC Press (2008);Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology 3^(rd) ed., revised ed., J. Wiley & Sons (New York, N. Y. 2006);Smith, March's Advanced Organic Chemistry Reactions, Mechanisms andStructure 7^(th) ed., J. Wiley & Sons (New York, N. Y. 2013); Singleton,Dictionary of DNA and Genome Technology 3^(rd) ed., Wiley-Blackwell(Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A LaboratoryManual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor,N. Y. 2012), provide one skilled in the art with a general guide to manyof the terms used in the present application. For references on how toprepare antibodies, see Greenfield, Antibodies A Laboratory Manual2^(nd) ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013);Köhler and Milstein, Derivation of specific antibody-producing tissueculture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul.,6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat. No.5,585,089 (1996 December); and Riechmann et al., Reshaping humanantibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Other features and advantages of theinvention will become apparent from the following detailed description,taken in conjunction with the accompanying drawings, which illustrate,by way of example, various features of embodiments of the invention.Indeed, the present invention is in no way limited to the methods andmaterials described. For convenience, certain terms employed herein, inthe specification, examples and appended claims are collected here.

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. The definitions are provided to aid indescribing particular embodiments, and are not intended to limit theclaimed invention, because the scope of the invention is limited only bythe claims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areuseful to an embodiment, yet open to the inclusion of unspecifiedelements, whether useful or not. It will be understood by those withinthe art that, in general, terms used herein are generally intended as“open” terms (e.g., the term “including” should be interpreted as“including but not limited to,” the term “having” should be interpretedas “having at least,” the term “includes” should be interpreted as“includes but is not limited to,” etc.).

Unless stated otherwise, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe application (especially in the context of claims) can be construedto cover both the singular and the plural. The recitation of ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate value falling within the range.Unless otherwise indicated herein, each individual value is incorporatedinto the specification as if it were individually recited herein. Allmethods described herein can be performed in any suitable order unlessotherwise indicated herein or otherwise clearly contradicted by context.The use of any and all examples, or exemplary language (for example,“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the application and does not pose alimitation on the scope of the application otherwise claimed. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.” No language in thespecification should be construed as indicating any non-claimed elementessential to the practice of the application.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” when used in reference to a disease, disorder or medicalcondition, refer to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent, reverse,alleviate, ameliorate, inhibit, lessen, slow down or stop theprogression or severity of a symptom or condition. The term “treating”includes reducing or alleviating at least one adverse effect or symptomof a condition. Treatment is generally “effective” if one or moresymptoms or clinical markers are reduced. Alternatively, treatment is“effective” if the progression of a disease, disorder or medicalcondition is reduced or halted. That is, “treatment” includes not justthe improvement of symptoms or markers, but also a cessation or at leastslowing of progress or worsening of symptoms that would be expected inthe absence of treatment. Also, “treatment” may mean to pursue or obtainbeneficial results, or lower the chances of the individual developingthe condition even if the treatment is ultimately unsuccessful. Those inneed of treatment include those already with the condition as well asthose prone to have the condition or those in whom the condition is tobe prevented.

“Beneficial results” or “desired results” may include, but are in no waylimited to, lessening or alleviating the severity of the diseasecondition, preventing the disease condition from worsening, curing thedisease condition, preventing the disease condition from developing,lowering the chances of a patient developing the disease condition,decreasing morbidity and mortality, and prolonging a patient's life orlife expectancy. As non-limiting examples, “beneficial results” or“desired results” may be alleviation of one or more symptom(s),diminishment of extent of the deficit, stabilized (i.e., not worsening)state of tumor, delay or slowing of tumor growth, and amelioration orpalliation of symptoms associated with tumor.

“Conditions” and “disease conditions,” as used herein may include, butare in no way limited to any form of malignant neoplastic cellproliferative disorders or diseases (e.g., tumor and cancer). Inaccordance with the present invention, “conditions” and “diseaseconditions,” as used herein include but are not limited to any and allconditions involving a tissue difference, i.e., normal vs. abnormal, dueto any and all reasons including but not limited to tumor, injury,trauma, ischemia, infection, inflammation, or auto-inflammation. Stillin accordance with the present invention, “conditions” and “diseaseconditions,” as used herein include but are not limited to any situationwhere a tissue of interest (e.g., a cancerous, injured, ischemic,infected, or inflammatory tissue) is different from the surroundingtissue (e.g., healthy tissues) due to physiological or pathologicalcauses. Examples of “conditions” and “disease conditions” include butare not limited to tumors, cancers, traumatic brain injury, spinal cordinjury, stroke, cerebral hemorrhage, brain ischemia, ischemic heartdiseases, ischemic reperfusion injury, cardiovascular diseases, heartvalve stenosis, infectious diseases, microbial infections, viralinfection, bacterial infection, fungal infection, and autoimmunediseases.

A “cancer” or “tumor” as used herein refers to an uncontrolled growth ofcells which interferes with the normal functioning of the bodily organsand systems, and/or all neoplastic cell growth and proliferation,whether malignant or benign, and all pre-cancerous and cancerous cellsand tissues. A subject that has a cancer or a tumor is a subject havingobjectively measurable cancer cells present in the subject's body.Included in this definition are benign and malignant cancers, as well asdormant tumors or micrometastasis. Cancers which migrate from theiroriginal location and seed vital organs can eventually lead to the deathof the subject through the functional deterioration of the affectedorgans. As used herein, the term “invasive” refers to the ability toinfiltrate and destroy surrounding tissue. Melanoma is an invasive formof skin tumor. As used herein, the term “carcinoma” refers to a cancerarising from epithelial cells. Examples of cancer include, but are notlimited to, nervous system tumor, brain tumor, nerve sheath tumor,breast cancer, colon cancer, carcinoma, lung cancer, hepatocellularcancer, gastric cancer, pancreatic cancer, cervical cancer, ovariancancer, liver cancer, bladder cancer, cancer of the urinary tract,thyroid cancer, renal cancer, renal cell carcinoma, carcinoma, melanoma,head and neck cancer, brain cancer, and prostate cancer, including butnot limited to androgen-dependent prostate cancer andandrogen-independent prostate cancer. Examples of brain tumor include,but are not limited to, benign brain tumor, malignant brain tumor,primary brain tumor, secondary brain tumor, metastatic brain tumor,glioma, glioblastoma multiforme (GBM), medulloblastoma, ependymoma,astrocytoma, pilocytic astrocytoma, oligodendroglioma, brainstem glioma,optic nerve glioma, mixed glioma such as oligoastrocytoma, low-gradeglioma, high-grade glioma, supratentorial glioma, infratentorial glioma,pontine glioma, meningioma, pituitary adenoma, and nerve sheath tumor.Nervous system tumor or nervous system neoplasm refers to any tumoraffecting the nervous system. A nervous system tumor can be a tumor inthe central nervous system (CNS), in the peripheral nervous system(PNS), or in both CNS and PNS. Examples of nervous system tumor includebut are not limited to brain tumor, nerve sheath tumor, and optic nerveglioma.

As used herein, the term “administering,” refers to the placement anagent as disclosed herein into a subject by a method or route whichresults in at least partial localization of the agents at a desiredsite. “Route of administration” may refer to any administration pathwayknown in the art, including but not limited to aerosol, nasal, oral,transmucosal, transdermal, parenteral, enteral, topical or local.“Parenteral” refers to a route of administration that is generallyassociated with injection, including intraorbital, infusion,intraarterial, intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal. Via the parenteral route, thecompositions may be in the form of solutions or suspensions for infusionor for injection, or as lyophilized powders. Via the enteral route, thepharmaceutical compositions can be in the form of tablets, gel capsules,sugar-coated tablets, syrups, suspensions, solutions, powders, granules,emulsions, microspheres or nanospheres or lipid vesicles or polymervesicles allowing controlled release.

The term “sample” or “biological sample” as used herein denotes aportion of a biological organism. The sample can be a cell, tissue,organ, or body part. A sample can still be integral of the biologicalorganism. For example, when a surgeon is trying to remove a breast tumorfrom a patient, the sample refers to the breast tissue labeled withinfrared dye and imaged with the imaging system described herein. Inthis situation, the sample is still part of the patient's body beforebeing removed. A sample can be taken or isolated from the biologicalorganism, e.g., a tumor sample removed from a subject. Exemplarybiological samples include, but are not limited to, a biofluid sample;serum; plasma; urine; saliva; a tumor sample; a tumor biopsy and/ortissue sample etc. The term also includes a mixture of theabove-mentioned samples. The term “sample” also includes untreated orpretreated (or pre-processed) biological samples. In some embodiments, asample can comprise one or more cells from the subject. In someembodiments, a sample can be a tumor cell sample, e.g. the sample cancomprise cancerous cells, cells from a tumor, and/or a tumor biopsy.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, and canine species, e.g., dog, fox, wolf. The terms,“patient”, “individual” and “subject” are used interchangeably herein.In an embodiment, the subject is mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. In addition, the methods described herein canbe used to treat domesticated animals and/or pets.

“Mammal” as used herein refers to any member of the class Mammalia,including, without limitation, humans and nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be included within the scope of this term.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g., tumor) or one or more complications related to the condition, andoptionally, have already undergone treatment for the condition or theone or more complications related to the condition. Alternatively, asubject can also be one who has not been previously diagnosed as havinga condition or one or more complications related to the condition. Forexample, a subject can be one who exhibits one or more risk factors fora condition or one or more complications related to the condition or asubject who does not exhibit risk factors. A “subject in need” oftreatment for a particular condition can be a subject suspected ofhaving that condition, diagnosed as having that condition, alreadytreated or being treated for that condition, not treated for thatcondition, or at risk of developing that condition.

The term “statistically significant” or “significantly” refers tostatistical evidence that there is a difference. It is defined as theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true. The decision is often made using thep-value.

In accordance with the invention, “channel”, “light channel”, and“optical channel” refer to a channel or pathway that conducts light fromone place to another. A “channel” can be an optical fiber, an opticalfilter, an optical enhancer, an optical attenuator, a beam splitter, acondenser, a diffuser, a collimating lens, a window, a hole, a mirror, ashutter, a lens or a set of lens, or a device including but not limitedto endoscope and microscope, or their various combinations.

In accordance with the invention, various infrared or near-infraredfluorophores may be used. Examples of these fluorophores include but arenot limited to various infrared or near-infrared fluorescent dyes andquantum dots. They are either alone or attached to a targeting moietysuch as a peptide, protein, nanoparticle, nanoconjugate, antibody, andnucleic acid (e.g., DNA and RNA strands) or to any other suchbiologically specific targeting entity. Near-infrared wavelength is aportion of infrared wavelength and is closest to the radiationdetectable by the human eye; and mid- and far-infrared are progressivelyfurther from the visible spectrum. As such, near-infrared fluorophoresare a subset of infrared fluorophores.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims.

Various embodiments of the present invention provide an imaging systemfor imaging a sample comprising an infrared or near-infraredfluorophore. The imaging system comprises: (1) a laser to emit anexcitation light for the infrared or near-infrared fluorophore, whereinthe excitation light is conducted to the sample; (2) a laser clean-upfilter in the excitation light path from the laser to the sample,wherein the laser clean-up filter narrows the wavelength band of theexcitation light to the peak absorption band of the infrared ornear-infrared fluorophore, wherein the narrowed excitation light excitesthe infrared or near-infrared fluorophore in the sample to emit anemission light, wherein the emission light is conducted to an imagesensor, and wherein there is no infrared filter in the emission lightpath from the sample to the image sensor; (3) a notch filter in theemission light path from the sample to the image sensor, wherein thenotch filter blocks the excitation light; and (4) a white light sourceto emit a light comprising visible light, wherein the visible light isconducted to the sample, wherein the sample reflects the visible light,wherein the reflected visible light is conducted to the image sensor,wherein the image sensor is one image sensor configured to detect boththe emission light and the visible light from the sample and configuredto generate sensor signals, and wherein the image sensor comprises blue,green and red pixel sensors.

In various embodiments, the emission light from the sample is not Ramanscattered light from the sample. In various embodiments, there is noFabry-Perot etalon, Raman analysis filter wheel, dispersive element,dispersive prism, isosceles prism, diffraction grating, reflection-typediffraction grating, or transmission-type diffraction grating in theemission light path from the sample to the image sensor. In variousembodiments, the emission light is not dispersed or filtered for Ramanband selection in the emission light path from the sample to the imagesensor. In various embodiments, the image sensor is configured not todetect Raman scattered light from the sample.

In various embodiments, the infrared or near-infrared fluorophore is onefrom the group consisting of: a cyanide-based infrared or near-infraredfluorophore, indocyanine green (ICG), a functional equivalent of ICG, ananalog of ICG, a derivative of ICG, a salt of ICG, IR800, Alexa680,cy5.5, a functional equivalent of IR800, a functional equivalent ofAlexa680, a functional equivalent of cy5.5, an analog of IR800, ananalog of Alexa680, an analog of cy5.5, a derivative of IR800, aderivative of Alexa680, a derivative of cy5.5, a salt of IR800, a saltof Alexa 680 or a salt of cy5.5. In some embodiments, the variousinfrared or near-infrared fluorophores described herein may be modifiedto be more or less lipophilic.

In various embodiments, the laser is pulsed. In various embodiments, thewhite light source is pulsed. In various embodiments, the image sensoris a CCD image sensor. In various embodiments, the image sensor is aCMOS image sensor. In various embodiments, the laser clean-up filter isnot a spatial filter. In various embodiments, the blocking range of thenotch filter is broader than the transmitting range of the laserclean-up filter.

In various embodiments, the excitation light comprises light having awavelength of about 785 nm. In various embodiments, the laser clean-upfilter selectively transmits light having a wavelength of about 785 nm.In various embodiments, the notch filter selectively blocks light havinga wavelength of about 785 nm.

In various embodiments, the imaging system further comprises a notchbeam splitter in the light path from the laser to the sample, wherebythe excitation light is reflected by the notch beam splitter to thesample. In various embodiments, the imaging system further comprises anotch beam splitter in the light path from the white light source to thesample, whereby the visible light is transmitted to the sample. Invarious embodiments, the imaging system further comprises a notch beamsplitter that reflects light having a wavelength of about 785 nm.

In various embodiments, the imaging system further comprises an imageprocessing unit to process sensor signals to generate image frames,wherein the image processing unit is connected to the image sensor. Invarious embodiments, the image processing unit process sensor signals togenerate at least one white light frame (WLF) when the sample receivesonly visible light, at least one stray light frame (SLF) when the samplereceives neither visible light nor the excitation light, and one or morenear infrared frames (NIFs) when the sample receives only excitationlight, and wherein the image processing unit subtracts the SLF from eachNIF and then adds together all SLF-subtracted NIFs to generate a finalNIF. In various embodiments, the image processing unit false colors thefinal NIF. In various embodiments, the image processing unit adds thefalse colored final NIF to the WLF to generate a composite image frameof visible light and infrared light.

In various embodiments, the imaging system further comprises an imagedisplaying unit to display images based on the image frames generatedfrom the image processing unit, wherein the image displaying unit isconnected to the image processing unit.

In various embodiments, the excitation light from the laser is conductedto the sample through a first channel, wherein the visible light fromthe white light source is conducted to the sample through a secondchannel, wherein the emission light emitted from the sample is conductedto the image sensor through a third channel, and wherein the visiblelight reflected from the sample is conducted to the image sensor througha fourth channel.

In various embodiments, the excitation light from the laser is conductedto the sample through a first light channel housed in an endoscope, thevisible light from the white light source is conducted to the samplethrough a second light channel housed in the endoscope; and the imagesensor is housed in the endoscope at or near the patient end of theendoscope. In some embodiments, the first light channel and the secondlight channel are one light channel. In other embodiments, the firstlight channel and the second light channel are two separate lightchannels. In various embodiments, the first light channel is an opticalcable. In various embodiments, the second light channel is an opticalcable. In various embodiments, the imaging system further comprises oneor more lenses in the emission light path and/or the visible light pathfrom the sample to the image sensor, wherein the one or more lenses arelocated at or near the patient end of the endoscope.

Various embodiments of the present invention provide a method forimaging a sample comprising an infrared or near-infrared fluorophore.The method comprises: (1) operating a laser to emit an excitation lightfor the infrared or near-infrared fluorophore; (2) conducting theexcitation light to the sample; (3) operating a laser clean-up filter inthe excitation light path from the laser to the sample to narrow thewavelength band of the excitation light to the peak absorption band ofthe infrared or near-infrared fluorophore, wherein the narrowedexcitation light excites the infrared or near-infrared fluorophore inthe sample to emit an emission light; (4) conducting the emission lightto an image sensor, wherein there is no infrared filter in the emissionlight path from the sample to the image sensor; (5) operating a notchfilter in the emission light path from the sample to the image sensor toblock the excitation light; (6) operating a white light source to emit alight comprising visible light; (7) conducting the visible light to thesample, wherein the sample reflects the visible light; (8) conductingthe reflected visible light to the image sensor; and (9) operating theimage sensor to detect both the emission light and the visible lightfrom the sample and to generate sensor signals, wherein the image sensoris one image sensor and comprises blue, green and red pixel sensors.

In various embodiments, the emission light from the sample is not Ramanscattered light from the sample. In various embodiments, the method doesnot include a step of operating Fabry-Perot etalon, Raman analysisfilter wheel, dispersive element, dispersive prism, isosceles prism,diffraction grating, reflection-type diffraction grating, ortransmission-type diffraction grating in the emission light path fromthe sample to the image sensor. In various embodiments, the method doesnot include a step of dispersing the emission light in the emissionlight path from the sample to the image sensor. In various embodiments,the method does not include a step of filtering the emission light forRaman band selection in the emission light path from the sample to theimage sensor. In various embodiments, the method does not include a stepof detecting Raman scatter light from the sample.

In various embodiments, the method further comprises performing asurgery on a subject to access the sample or to isolate the sample. Invarious embodiments, the method further comprises labeling the samplewith an infrared or near-infrared fluorophore.

In various embodiments, the present invention provides an imaging systemfor imaging a sample. In accordance with the invention, the samplecomprises an infrared or near-infrared fluorophore. The imaging systemcomprises: an image sensor, a laser, a laser clean-up filter, a notchfilter, and a white light source. The image sensor detects visible lightand infrared light and to generate sensor signals. The laser emits anexcitation light for the infrared or near-infrared fluorophore. Thelaser clean-up filter is placed in the light path from the laser to thesample, and narrows the wavelength band of the excitation light to thepeak absorption band of the infrared or near-infrared fluorophore. Thenarrowed excitation light excites the infrared or near-infraredfluorophore in the sample to emit an emission light. The notch filter isplaced in the light path from the sample to the image sensor, and blocksthe excitation light. The white light source emits a light comprisingvisible light. In accordance with the invention, visible light can havea spectrum of 400-700 nm. In various embodiments, the imaging systemfurther comprises a fast trigger unit.

In some embodiments, there is an infrared filter in the light path fromthe white light source to the sample. In various embodiments, theintensity of the laser is controlled to ensure uniform excitation on thesame area illuminated by visible light. Although lasers by definitionare monochromatic, which mean they do not have a broad band range, inpractice most lasers will have a small amount of emission in theadjacent color bands. In various embodiments, the laser is a narrow bandlaser including but not limited to a laser having a wavelength rangethat spans no more than 5, 10, 15, or 20 nm. As a non-limiting example,the laser can emit light having about 775-795 nm wavelength with a peakat about 785 nm (FIG. 7).

In various embodiments, the blocking range of the notch filter isbroader than the transmitting range of the laser clean-up filter. Invarious embodiments, the blocking range of the notch filter is about5-10 nm, 10-15 nm, or 15-20 nm broader than the transmitting range ofthe laser clean-up filter. In various embodiments, the blocking range ofthe notch filter is about 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-40%,40-50%, 50-100% or 100-200% broader than the transmitting range of thelaser clean-up filter. As a non-limiting example, the transmitting rangeof the laser clean-up filter can be about 775-795 nm and the blockingrange of the notch filter can be about 770-800 nm, 765-805 nm, or760-810 nm.

In various embodiments, the excitation light comprises light having awavelength of about 785 nm. In various embodiments, the laser clean-upfilter selectively transmits light having a wavelength of about 785 nm.In various embodiments, the notch filter selectively blocks light havinga wavelength of about 785 nm.

In various embodiments, the imaging system further comprises a notchbeam splitter in the light path from the laser to the sample, wherebythe excitation light is reflected by the notch beam splitter to thesample. In various embodiments, the imaging system further comprises anotch beam splitter in the light path from the white light source to thesample, whereby the visible light is transmitted to the sample. Thenotch beam splitter in the light path from the laser to the sample andthe notch beam splitter in the light path from the white light source tothe sample can be one single notch beam splitter or two separate notchbeam splitters. In one embodiment, the notch beam splitter can splitlight at a wavelength of about 700, 725 or 750 nm. In anotherembodiment, the notch beam splitter that reflects light having awavelength of about 785 nm.

In various embodiments, there is no infrared filter in the light pathfrom the sample to the image sensor. In various embodiments, there is noinfrared filter in the light path from the laser to the sample. In someembodiments, there is an optical filter to block the excitation light inthe light path from the sample to the image sensor. In otherembodiments, there is no optical filter to block the excitation light inthe light path from the laser to the sample.

In various embodiments, the imaging system further comprises an imageprocessing unit to process sensor signals to generate image frames. Inaccordance with the present invention, the image processing unit isconnected to the image sensor. In various embodiments, the imageprocessing unit process sensor signals to generate at least one whitelight frame (WLF) when the sample receives only visible light, at leastone stray light frame (SLF) when the sample receives neither visiblelight nor the excitation light, and one or more near infrared frames(NIFs) when the sample receives only excitation light, and wherein theimage processing unit subtracts the SLF from each NIF and then addstogether all SLF-subtracted NIFs to generate a final NIF. In variousembodiments, the image processing unit false colors the final NIF. Invarious embodiments, the image processing unit adds the false coloredfinal NIF to the WLF to generate a composite image frame of visiblelight and infrared light. In various embodiments, the image processingunit generates composite image frames of visible light and infraredlight at a frequency of 30 Hz.

In various embodiments, during one cycle of generating one compositeimage frame of visible light and infrared light, the imaging systemgenerates one or more WLFs, one or more SLFs, and one or more NIFs. Inaccordance with the present invention, the sequence of WLF (W), SLF (S)and NIF (N) during one cycle has many suitable choices, including butnot limited to, W-S-N, W-N-S, S-W-N, S-N-W, N-S-W, and N-W-S. Still inaccordance with the present invention, the numbers of WLF (W), SLF (S)and NIF (N) during one cycle has many suitable choices, including butnot limited to, 1W-1S-1N, 1W-1S-2N, 1W-1S-3N, 2W-2S-6N, and1W-1S-3N-1W-1S-3N. In various embodiments, the imaging systemcontinuously repeats a cycle to generate a continuous stream ofcomposite image frames as a real-time video.

In various embodiments, the imaging system further comprises an imagedisplaying unit to display images based on the image frames generatedfrom the image processing unit. In accordance with the presentinvention, the image displaying unit is connected to the imageprocessing unit. Examples of the image displaying unit include but arenot limited to monitors, projectors, phones, tablets, and screens. Insome embodiments, the image displaying unit displays composite imageframes of visible light and infrared light at a frequency of 30 Hz.

In various embodiments, the imaging system further comprises a firstchannel to conduct the excitation light from the laser to the sample, asecond channel to conduct the visible light from the white light sourceto the sample, a third channel to conduct the emission light from thesample to the image sensor, and a fourth channel to conduct the visiblelight from the sample to the image sensor. In accordance with thepresent invention, the first, second, third and fourth channels are fourseparate channels or combined into one, two, or three channels. Still inaccordance with the present invention, two or more of the four channelsmay overlap partially or completely on their light paths. In variousembodiments, the first, second, third and fourth channels are endoscopeor microscope.

In various embodiments, the present invention provides an imaging systemfor imaging a sample. In accordance with the invention, the samplecomprises an infrared or near-infrared fluorophore. As a non-limitingexample, the infrared or near-infrared fluorophore can be acyanide-based infrared or near-infrared fluorophore (e.g., indocyaninegreen (ICG)). The system comprises: (a) an image sensor, (b) a laser,(c) a laser clean-up filter, (d) a first channel, (e) a white lightsource, (f) a second channel, (g) a notch beam splitter, (h) a thirdchannel, (i) a fourth channel, (j) a notch filter, (k) an imageprocessing unit, and (1) an image displaying unit. (a) The image sensordetects visible light and infrared light and generates sensor signals ata first frequency. There is no infrared filter in the light path fromthe sample to the image sensor. The image sensor comprises blue, greenand red pixel sensors. Examples of the image sensor include but are notlimited to CCD image sensors and CMOS image sensors. (b) The laser emitsan excitation light for the infrared or near-infrared fluorophore. (c)The laser clean-up filter is placed in the light path from the laser tothe sample. The laser clean-up filter narrows the wavelength band of theexcitation light to the peak absorption band of the infrared ornear-infrared fluorophore, and the narrowed excitation light excites theinfrared or near-infrared fluorophore in the sample to emit an emissionlight. (d) The first channel conducts the excitation light from thelaser to the sample. (e) The white light source emits a light comprisingvisible light. (f) The second channel conducts the visible light fromthe white light source to the sample. (g) The notch beam splitter isplaced in the light path from the laser to the sample and in the lightpath from the white light source to the sample. The excitation light isreflected by the notch beam splitter to the sample and the visible lightis transmitted through the notch beam splitter to the sample. (h) Thethird channel conducts the emission light from the sample to the imagesensor. (i) The fourth channel conducts the visible light from thesample to the image sensor. (j) The notch filter is placed in the lightpath from the sample to the image sensor, and the notch filter blocksthe excitation light. (k) The image processing unit is connected to theimage sensor and processes sensor signals to generate image frames. Atleast one white light frame (WLF) is generated when the sample receivesonly visible light, at least one stray light frame (SLF) is generatedwhen the sample receives neither visible light nor the excitation light,and one or more near infrared frames (NIFs) are generated when thesample receives only excitation light. The image processing unitsubtracts the SLF from each NIF and then adds together allSLF-subtracted NIFs to generate a final NIF. The image processing unitfalse colors the final NIF and adds the false colored final NIF to theWLF to generate a composite image frame of visible light and infraredlight. (1) The image displaying unit is connected to the imageprocessing unit and displays images based on the image frames generatedfrom the image processing unit.

In various embodiments, the present invention provides an imaging systemfor imaging a sample. In accordance with the invention, the samplecomprises an infrared or near-infrared fluorophore. As a non-limitingexample, the infrared or near-infrared fluorophore can be acyanide-based infrared or near-infrared fluorophore (e.g., indocyaninegreen (ICG)). The system comprises: (a) a laser; (b) a white lightsource; (c) an endoscope comprising a first light channel, a secondlight channel, an image sensor and a notch filter; (d) an imageprocessing unit; and (e) an image displaying unit. (a) The laser emitsan excitation light for the infrared or near-infrared fluorophore. (b)The white light source emits a light comprising visible light. (c) Thefirst light channel conducts the excitation light from the laser to thesample; the second channel conducts the visible light from the whitelight source to the sample; the image sensor is located at or near thepatient end of the endoscope, and detects visible light and infraredlight and generates sensor signals; and the notch filter is located ator near the patient end of the endoscope, is in the light path from thesample to the image sensor, and blocks the excitation light. (d) Theimage processing unit is connected to the image sensor, and processessensor signals to generate image frames. (e) The image displaying unitis connected to the image processing unit, and displays images based onthe image frames generated form the image processing unit. In someembodiments, the laser is pulsed. In some embodiments, the white lightsource is pulsed. In some embodiments, the first light channel is anoptical cable. In some embodiments, the second light channel is anoptical cable. In some embodiments, the image processing unit isconnected to the image sensor through an electrical cable. In variousembodiments, the imaging system further comprises one or more lenses inthe light path from the sample to the image sensor, wherein the one ormore lenses are located at or near the patient end of the endoscope.

In various embodiments, the image sensor comprises blue, green and redpixel sensors. In one embodiment, all the blue, green and red pixelsensors are sensitive to both visible light and infrared light. Invarious embodiments, the image sensor is a CCD image sensor that detectsvisible light and infrared light and generates CCD image signals. Invarious embodiments, the image sensor is a CMOS image sensor thatdetects visible light and infrared light and generates CMOS imagesignals. In various embodiments, the image sensor is without a NIR longpass filter.

In various embodiments, the imaging system further comprises softwarethat controls all the components of the imaging system. FIG. 9 depicts adevice or a computer system 900 comprising one or more processors 930and a memory 940 storing one or more programs 950 for execution by theone or more processors 930.

In some embodiments, the device or computer system 900 can furthercomprise a non-transitory computer-readable storage medium 960 storingthe one or more programs 950 for execution by the one or more processors930 of the device or computer system 900.

In some embodiments, the device or computer system 900 can furthercomprise one or more input devices 910, which can be configured to sendor receive information to or from any one from the group consisting of:an external device (not shown), the one or more processors 930, thememory 940, the non-transitory computer-readable storage medium 960, andone or more output devices 970. The one or more input devices 910 can beconfigured to wirelessly send or receive information to or from theexternal device via a means for wireless communication, such as anantenna 920, a transceiver (not shown) or the like.

In some embodiments, the device or computer system 900 can furthercomprise one or more output devices 970, which can be configured to sendor receive information to or from any one from the group consisting of:an external device (not shown), the one or more input devices 910, theone or more processors 930, the memory 940, and the non-transitorycomputer-readable storage medium 960. The one or more output devices 970can be configured to wirelessly send or receive information to or fromthe external device via a means for wireless communication, such as anantenna 980, a transceiver (not shown) or the like.

Each of the above identified modules or programs correspond to a set ofinstructions for performing a function described above. These modulesand programs (i.e., sets of instructions) need not be implemented asseparate software programs, procedures or modules, and thus varioussubsets of these modules may be combined or otherwise re-arranged invarious embodiments. In some embodiments, memory may store a subset ofthe modules and data structures identified above. Furthermore, memorymay store additional modules and data structures not described above.

The illustrated aspects of the disclosure may also be practiced indistributed computing environments where certain tasks are performed byremote processing devices that are linked through a communicationsnetwork. In a distributed computing environment, program modules can belocated in both local and remote memory storage devices.

Moreover, it is to be appreciated that various components describedherein can include electrical circuit(s) that can include components andcircuitry elements of suitable value in order to implement theembodiments of the subject innovation(s). Furthermore, it can beappreciated that many of the various components can be implemented onone or more integrated circuit (IC) chips. For example, in oneembodiment, a set of components can be implemented in a single IC chip.In other embodiments, one or more of respective components arefabricated or implemented on separate IC chips.

What has been described above includes examples of the embodiments ofthe present invention. It is, of course, not possible to describe everyconceivable combination of components or methodologies for purposes ofdescribing the claimed subject matter, but it is to be appreciated thatmany further combinations and permutations of the subject innovation arepossible. Accordingly, the claimed subject matter is intended to embraceall such alterations, modifications, and variations that fall within thespirit and scope of the appended claims. Moreover, the above descriptionof illustrated embodiments of the subject disclosure, including what isdescribed in the Abstract, is not intended to be exhaustive or to limitthe disclosed embodiments to the precise forms disclosed. While specificembodiments and examples are described herein for illustrative purposes,various modifications are possible that are considered within the scopeof such embodiments and examples, as those skilled in the relevant artcan recognize.

In particular and in regard to the various functions performed by theabove described components, devices, circuits, systems and the like, theterms used to describe such components are intended to correspond,unless otherwise indicated, to any component which performs thespecified function of the described component (e.g., a functionalequivalent), even though not structurally equivalent to the disclosedstructure, which performs the function in the herein illustratedexemplary aspects of the claimed subject matter. In this regard, it willalso be recognized that the innovation includes a system as well as acomputer-readable storage medium having computer-executable instructionsfor performing the acts and/or events of the various methods of theclaimed subject matter.

The aforementioned systems/circuits/modules have been described withrespect to interaction between several components/blocks. It can beappreciated that such systems/circuits and components/blocks can includethose components or specified sub-components, some of the specifiedcomponents or sub-components, and/or additional components, andaccording to various permutations and combinations of the foregoing.Sub-components can also be implemented as components communicativelycoupled to other components rather than included within parentcomponents (hierarchical). Additionally, it should be noted that one ormore components may be combined into a single component providingaggregate functionality or divided into several separate sub-components,and any one or more middle layers, such as a management layer, may beprovided to communicatively couple to such sub-components in order toprovide integrated functionality. Any components described herein mayalso interact with one or more other components not specificallydescribed herein but known by those of skill in the art.

In addition, while a particular feature of the subject innovation mayhave been disclosed with respect to only one of several implementations,such feature may be combined with one or more other features of theother implementations as may be desired and advantageous for any givenor particular application. Furthermore, to the extent that the terms“includes,” “including,” “has,” “contains,” variants thereof, and othersimilar words are used in either the detailed description or the claims,these terms are intended to be inclusive in a manner similar to the term“comprising” as an open transition word without precluding anyadditional or other elements.

As used in this application, the terms “component,” “module,” “system,”or the like are generally intended to refer to a computer-relatedentity, either hardware (e.g., a circuit), a combination of hardware andsoftware, software, or an entity related to an operational machine withone or more specific functionalities. For example, a component may be,but is not limited to being, a process running on a processor (e.g.,digital signal processor), a processor, an object, an executable, athread of execution, a program, and/or a computer. By way ofillustration, both an application running on a controller and thecontroller can be a component. One or more components may reside withina process and/or thread of execution and a component may be localized onone computer and/or distributed between two or more computers. Further,a “device” can come in the form of specially designed hardware;generalized hardware made specialized by the execution of softwarethereon that enables the hardware to perform specific function; softwarestored on a computer-readable medium; or a combination thereof.

Moreover, the words “example” or “exemplary” are used herein to meanserving as an example, instance, or illustration. Any aspect or designdescribed herein as “exemplary” is not necessarily to be construed aspreferred or advantageous over other aspects or designs. Rather, use ofthe words “example” or “exemplary” is intended to present concepts in aconcrete fashion. As used in this application, the term “or” is intendedto mean an inclusive “or” rather than an exclusive “or”. That is, unlessspecified otherwise, or clear from context, “X employs A or B” isintended to mean any of the natural inclusive permutations. That is, ifX employs A; X employs B; or X employs both A and B, then “X employs Aor B” is satisfied under any of the foregoing instances. In addition,the articles “a” and “an” as used in this application and the appendedclaims should generally be construed to mean “one or more” unlessspecified otherwise or clear from context to be directed to a singularform.

Computing devices typically include a variety of media, which caninclude computer-readable storage media and/or communications media, inwhich these two terms are used herein differently from one another asfollows. Computer-readable storage media can be any available storagemedia that can be accessed by the computer, is typically of anon-transitory nature, and can include both volatile and nonvolatilemedia, removable and non-removable media. By way of example, and notlimitation, computer-readable storage media can be implemented inconnection with any method or technology for storage of information suchas computer-readable instructions, program modules, structured data, orunstructured data. Computer-readable storage media can include, but arenot limited to, RAM, ROM, EEPROM, flash memory or other memorytechnology, CD-ROM, digital versatile disk (DVD) or other optical diskstorage, magnetic cassettes, magnetic tape, magnetic disk storage orother magnetic storage devices, or other tangible and/or non-transitorymedia which can be used to store desired information. Computer-readablestorage media can be accessed by one or more local or remote computingdevices, e.g., via access requests, queries or other data retrievalprotocols, for a variety of operations with respect to the informationstored by the medium.

On the other hand, communications media typically embodycomputer-readable instructions, data structures, program modules orother structured or unstructured data in a data signal that can betransitory such as a modulated data signal, e.g., a carrier wave orother transport mechanism, and includes any information delivery ortransport media. The term “modulated data signal” or signals refers to asignal that has one or more of its characteristics set or changed insuch a manner as to encode information in one or more signals. By way ofexample, and not limitation, communication media include wired media,such as a wired network or direct-wired connection, and wireless mediasuch as acoustic, RF, infrared and other wireless media.

In view of the exemplary systems described above, methodologies that maybe implemented in accordance with the described subject matter will bebetter appreciated with reference to the flowcharts of the variousfigures. For simplicity of explanation, the methodologies are depictedand described as a series of acts. However, acts in accordance with thisdisclosure can occur in various orders and/or concurrently, and withother acts not presented and described herein. Furthermore, not allillustrated acts may be required to implement the methodologies inaccordance with the disclosed subject matter. In addition, those skilledin the art will understand and appreciate that the methodologies couldalternatively be represented as a series of interrelated states via astate diagram or events. Additionally, it should be appreciated that themethodologies disclosed in this specification are capable of beingstored on an article of manufacture to facilitate transporting andtransferring such methodologies to computing devices. The term articleof manufacture, as used herein, is intended to encompass a computerprogram accessible from any computer-readable device or storage media.

In various embodiments, the present invention provides a computerimplemented method for imaging a sample comprising an infrared ornear-infrared fluorophore, comprising: on a device having one or moreprocessors and a memory storing one or more programs for execution bythe one or more processors, the one or more programs includinginstructions for: operating an image sensor to detect visible light andinfrared light and generating sensor signals; operating a laser to emitan excitation light for the infrared or near-infrared fluorophore;operating a laser clean-up filter in the light path from the laser tothe sample, whereby the laser clean-up filter narrows the wavelengthband of the excitation light to the peak absorption band of the infraredor near-infrared fluorophore, and whereby the narrowed excitation lightexcites the infrared or near-infrared fluorophore in the sample to emitan emission light; operating a notch filter in the light path from thesample to the image sensor, whereby the notch filter blocks theexcitation light; and operating a white light source to emit a lightcomprising visible light.

In various embodiments, the present invention provides a computer systemfor imaging a sample comprising an infrared or near-infraredfluorophore, comprising: one or more processors and memory to store oneor more programs, the one or more programs comprising instructions for:operating an image sensor to detect visible light and infrared light andgenerating sensor signals; operating a laser to emit an excitation lightfor the infrared or near-infrared fluorophore; operating a laserclean-up filter in the light path from the laser to the sample, wherebythe laser clean-up filter narrows the wavelength band of the excitationlight to the peak absorption band of the infrared or near-infraredfluorophore, and whereby the narrowed excitation light excites theinfrared or near-infrared fluorophore in the sample to emit an emissionlight; operating a notch filter in the light path from the sample to theimage sensor, whereby the notch filter blocks the excitation light; andoperating a white light source to emit a light comprising visible light.

In various embodiments, the present invention provides a non-transitorycomputer-readable storage medium storing one or more programs forimaging a sample comprising an infrared or near-infrared fluorophore,the one or more programs for execution by one or more processors of acomputer system, the one or more programs comprising instructions for:operating an image sensor to detect visible light and infrared light andgenerating sensor signals; operating a laser to emit an excitation lightfor the infrared or near-infrared fluorophore; operating a laserclean-up filter in the light path from the laser to the sample, wherebythe laser clean-up filter narrows the wavelength band of the excitationlight to the peak absorption band of the infrared or near-infraredfluorophore, and whereby the narrowed excitation light excites theinfrared or near-infrared fluorophore in the sample to emit an emissionlight; operating a notch filter in the light path from the sample to theimage sensor, whereby the notch filter blocks the excitation light; andoperating a white light source to emit a light comprising visible light.

In various embodiments, the present invention provides a computerimplemented method for imaging a sample comprising an infrared ornear-infrared fluorophore, comprising: on a device having one or moreprocessors and a memory storing one or more programs for execution bythe one or more processors, the one or more programs includinginstructions for: (a) operating an image sensor to detect visible lightand infrared light and generate sensor signals, wherein there is noinfrared filter in the light path from the sample to the image sensor,and wherein the image sensor comprises blue, green and red pixelsensors; (b) operating a laser to emit an excitation light for theinfrared or near-infrared fluorophore; (c) operating a laser clean-upfilter in the light path from the laser to the sample, whereby the laserclean-up filter narrows the wavelength band of the excitation light tothe peak absorption band of the infrared or near-infrared fluorophore,and whereby the narrowed excitation light excites the infrared ornear-infrared fluorophore in the sample to emit an emission light; (d)operating a first channel to conduct the excitation light from the laserto the sample; (e) operating a white light source to emit a lightcomprising visible light; (f) operating a second channel to conduct thevisible light from the white light source to the sample; (g) operating anotch beam splitter in the light path from the laser to the sample andin the light path from the white light source to the sample, whereby theexcitation light is reflected by the notch beam splitter to the sampleand the visible light is transmitted through the notch beam splitter tothe sample; (h) operating a third channel to conduct the emission lightfrom the sample to the image sensor; (i) operating a fourth channel toconduct the visible light from the sample to the image sensor; (j)operating a notch filter in the light path from the sample to the imagesensor, whereby the notch filter blocks the excitation light; and (k)operating an image processing unit to process sensor signals to generateimage frames, wherein the image processing unit is connected to theimage sensor, wherein at least one white light frame (WLF) is generatedwhen the sample receives only visible light, wherein at least one straylight frame (SLF) is generated when the sample receives neither visiblelight nor the excitation light, wherein one or more near infrared frames(NIFs) are generated when the sample receives only excitation light,wherein the image processing unit subtracts the SLF from each NIF andthen adds together all SLF-subtracted NIFs to generate a final NIF,wherein the image processing unit false colors the final NIF, andwherein the image processing unit adds the false colored final NIF tothe WLF to generate a composite image frame of visible light andinfrared light. (1) operating an image displaying unit to display imagesbased on the image frames generated from the image processing unit,wherein the image displaying unit is connected to the image processingunit.

In various embodiments, the present invention provides a computer systemfor imaging a sample comprising an infrared or near-infraredfluorophore, comprising: one or more processors and memory to store oneor more programs, the one or more programs comprising instructions for:(a) operating an image sensor to detect visible light and infrared lightand generate sensor signals, wherein there is no infrared filter in thelight path from the sample to the image sensor, and wherein the imagesensor comprises blue, green and red pixel sensors; (b) operating alaser to emit an excitation light for the infrared or near-infraredfluorophore; (c) operating a laser clean-up filter in the light pathfrom the laser to the sample, whereby the laser clean-up filter narrowsthe wavelength band of the excitation light to the peak absorption bandof the infrared or near-infrared fluorophore, and whereby the narrowedexcitation light excites the infrared or near-infrared fluorophore inthe sample to emit an emission light; (d) operating a first channel toconduct the excitation light from the laser to the sample; (e) operatinga white light source to emit a light comprising visible light; (f)operating a second channel to conduct the visible light from the whitelight source to the sample; (g) operating a notch beam splitter in thelight path from the laser to the sample and in the light path from thewhite light source to the sample, whereby the excitation light isreflected by the notch beam splitter to the sample and the visible lightis transmitted through the notch beam splitter to the sample; (h)operating a third channel to conduct the emission light from the sampleto the image sensor; (i) operating a fourth channel to conduct thevisible light from the sample to the image sensor; (j) operating a notchfilter in the light path from the sample to the image sensor, wherebythe notch filter blocks the excitation light; (k) operating an imageprocessing unit to process sensor signals to generate image frames,wherein the image processing unit is connected to the image sensor,wherein at least one white light frame (WLF) is generated when thesample receives only visible light, wherein at least one stray lightframe (SLF) is generated when the sample receives neither visible lightnor the excitation light, wherein one or more near infrared frames(NIFs) are generated when the sample receives only excitation light,wherein the image processing unit subtracts the SLF from each NIF andthen adds together all SLF-subtracted NIFs to generate a final NIF,wherein the image processing unit false colors the final NIF, andwherein the image processing unit adds the false colored final NIF tothe WLF to generate a composite image frame of visible light andinfrared light; and (1) operating an image displaying unit to displayimages based on the image frames generated from the image processingunit, wherein the image displaying unit is connected to the imageprocessing unit.

In various embodiments, the present invention provides a non-transitorycomputer-readable storage medium storing one or more programs forimaging a sample comprising an infrared or near-infrared fluorophore,the one or more programs for execution by one or more processors of acomputer system, the one or more programs comprising instructions for:(a) operating an image sensor to detect visible light and infrared lightand generate sensor signals, wherein there is no infrared filter in thelight path from the sample to the image sensor, and wherein the imagesensor comprises blue, green and red pixel sensors; (b) operating alaser to emit an excitation light for the infrared or near-infraredfluorophore; (c) operating a laser clean-up filter in the light pathfrom the laser to the sample, whereby the laser clean-up filter narrowsthe wavelength band of the excitation light to the peak absorption bandof the infrared or near-infrared fluorophore, and whereby the narrowedexcitation light excites the infrared or near-infrared fluorophore inthe sample to emit an emission light; (d) operating a first channel toconduct the excitation light from the laser to the sample; (e) operatinga white light source to emit a light comprising visible light; (f)operating a second channel to conduct the visible light from the whitelight source to the sample; (g) operating a notch beam splitter in thelight path from the laser to the sample and in the light path from thewhite light source to the sample, whereby the excitation light isreflected by the notch beam splitter to the sample and the visible lightis transmitted through the notch beam splitter to the sample; (h)operating a third channel to conduct the emission light from the sampleto the image sensor; (i) operating a fourth channel to conduct thevisible light from the sample to the image sensor; (j) operating a notchfilter in the light path from the sample to the image sensor, wherebythe notch filter blocks the excitation light; (k) operating an imageprocessing unit to process sensor signals to generate image frames,wherein the image processing unit is connected to the image sensor,wherein at least one white light frame (WLF) is generated when thesample receives only visible light, wherein at least one stray lightframe (SLF) is generated when the sample receives neither visible lightnor the excitation light, wherein one or more near infrared frames(NIFs) are generated when the sample receives only excitation light,wherein the image processing unit subtracts the SLF from each NIF andthen adds together all SLF-subtracted NIFs to generate a final NIF,wherein the image processing unit false colors the final NIF, andwherein the image processing unit adds the false colored final NIF tothe WLF to generate a composite image frame of visible light andinfrared light; and (1) operating an image displaying unit to displayimages based on the image frames generated from the image processingunit, wherein the image displaying unit is connected to the imageprocessing unit.

In various embodiments, the present invention provides a computerimplemented method for imaging a sample comprising an infrared ornear-infrared fluorophore, comprising: on a device having one or moreprocessors and a memory storing one or more programs for execution bythe one or more processors, the one or more programs includinginstructions for: operating an image sensor to detect visible light andinfrared light and generate sensor signals; operating a laser to emit anexcitation light for the infrared or near-infrared fluorophore andalternate between on and off statuses; operating a notch beam splitterin the light path from the laser to the sample and in the light pathfrom the sample to the image sensor, whereby the excitation light isreflected by the notch beam splitter to the sample, whereby theexcitation light excites the infrared or near-infrared fluorophore inthe sample to emit an emission light, and whereby the emission light istransmitted through the notch beam splitter to the image sensor;operating a notch filter in the light path from the sample to the imagesensor, whereby the notch filter blocks the excitation light; andoperating a synchronization module to synchronize the image sensor withthe laser and visible light, whereby a single sensor signal issynchronized to a single on or off status of the laser.

In various embodiments, the present invention provides a computer systemfor imaging a sample comprising an infrared or near-infraredfluorophore, comprising: one or more processors and memory to store oneor more programs, the one or more programs comprising instructions for:operating an image sensor to detect visible light and infrared light andgenerate sensor signals; operating a laser to emit an excitation lightfor the infrared or near-infrared fluorophore and alternate between onand off statuses; operating a notch beam splitter in the light path fromthe laser to the sample and in the light path from the sample to theimage sensor, whereby the excitation light is reflected by the notchbeam splitter to the sample, whereby the excitation light excites theinfrared or near-infrared fluorophore in the sample to emit an emissionlight, and whereby the emission light is transmitted through the notchbeam splitter to the image sensor; operating a notch filter in the lightpath from the sample to the image sensor, whereby the notch filterblocks the excitation light; and operating a synchronization module tosynchronize the image sensor with the laser and visible light, whereby asingle sensor signal is synchronized to a single on or off status of thelaser.

In various embodiments, the present invention provides a non-transitorycomputer-readable storage medium storing one or more programs forimaging a sample comprising an infrared or near-infrared fluorophore,the one or more programs for execution by one or more processors of acomputer system, the one or more programs comprising instructions for:operating an image sensor to detect visible light and infrared light andgenerate sensor signals; operating a laser to emit an excitation lightfor the infrared or near-infrared fluorophore and alternate between onand off statuses; operating a notch beam splitter in the light path fromthe laser to the sample and in the light path from the sample to theimage sensor, whereby the excitation light is reflected by the notchbeam splitter to the sample, whereby the excitation light excites theinfrared or near-infrared fluorophore in the sample to emit an emissionlight, and whereby the emission light is transmitted through the notchbeam splitter to the image sensor; operating a notch filter in the lightpath from the sample to the image sensor, whereby the notch filterblocks the excitation light; and operating a synchronization module tosynchronize the image sensor with the laser and visible light, whereby asingle sensor signal is synchronized to a single on or off status of thelaser.

In various embodiments, the present invention provides a computerimplemented method for imaging a sample comprising an infrared ornear-infrared fluorophore, comprising: on a device having one or moreprocessors and a memory storing one or more programs for execution bythe one or more processors, the one or more programs includinginstructions for: (a) operating an image sensor to detect visible lightand infrared light and generate sensor signals at a first frequency,wherein there is no infrared filter in the light path from the sample tothe image sensor, and wherein the image sensor comprises blue, green andred pixel sensors; (b) operating a laser to emit an excitation light forthe infrared or near-infrared fluorophore and to alternate between onand off statuses at a second frequency, wherein the second frequency ishalf of the first frequency; (c) operating a first channel to conductthe excitation light from the laser to the sample; (d) operating a lightsource to emit a light comprising visible light; (e) operating a secondchannel to conduct the visible light from the light source to thesample; (f) operating a notch beam splitter in the light path from thelaser to the sample and in the light path from the sample to the imagesensor, whereby the excitation light is reflected by the notch beamsplitter to the sample, whereby the excitation light excites theinfrared or near-infrared fluorophore in the sample to emit an emissionlight, and whereby the emission light is transmitted through the notchbeam splitter to the image sensor; (g) operating a third channel toconduct the emission light from the sample to the image sensor; (h)operating a fourth channel to conduct the visible light from the sampleto the image sensor; (i) operating a notch filter in the light path fromthe sample to the image sensor, whereby the notch filter blocks theexcitation light; (j) operating a synchronization module to synchronizethe image sensor with the laser and visible light, whereby a singlesensor signal is synchronized to a single on or off status of the laser;(k) operating an image processing unit to process sensor signals togenerate image frames, wherein the image processing unit is connected tothe image sensor, wherein the image processing unit subtracts an imageframe generated when the laser is off from the previous or next imageframe generated when the laser is on, whereby an infrared-only imageframe is generated upon the difference between the two successive imageframes, wherein the image processing unit false colors the infrared-onlyimage frame, wherein the image processing unit adds the false coloredinfrared-only image frame back to the image frame generated when thelaser is off, whereby a composite image frame of visible light andinfrared light is generated; and (1) operating an image displaying unitto display images based on the image frames generated from the imageprocessing unit, wherein the image displaying unit is connected to theimage processing unit.

In various embodiments, the present invention provides a computer systemfor imaging a sample comprising an infrared or near-infraredfluorophore, comprising: one or more processors and memory to store oneor more programs, the one or more programs comprising instructions for:(a) operating an image sensor to detect visible light and infrared lightand generate sensor signals at a first frequency, wherein there is noinfrared filter in the light path from the sample to the image sensor,and wherein the image sensor comprises blue, green and red pixelsensors; (b) operating a laser to emit an excitation light for theinfrared or near-infrared fluorophore and to alternate between on andoff statuses at a second frequency, wherein the second frequency is halfof the first frequency; (c) operating a first channel to conduct theexcitation light from the laser to the sample; (d) operating a lightsource to emit a light comprising visible light; (e) operating a secondchannel to conduct the visible light from the light source to thesample; (f) operating a notch beam splitter in the light path from thelaser to the sample and in the light path from the sample to the imagesensor, whereby the excitation light is reflected by the notch beamsplitter to the sample, whereby the excitation light excites theinfrared or near-infrared fluorophore in the sample to emit an emissionlight, and whereby the emission light is transmitted through the notchbeam splitter to the image sensor; (g) operating a third channel toconduct the emission light from the sample to the image sensor; (h)operating a fourth channel to conduct the visible light from the sampleto the image sensor; (i) operating a notch filter in the light path fromthe sample to the image sensor, whereby the notch filter blocks theexcitation light; (j) operating a synchronization module to synchronizethe image sensor with the laser and visible light, whereby a singlesensor signal is synchronized to a single on or off status of the laser;(k) operating an image processing unit to process sensor signals togenerate image frames, wherein the image processing unit is connected tothe image sensor, wherein the image processing unit subtracts an imageframe generated when the laser is off from the previous or next imageframe generated when the laser is on, whereby an infrared-only imageframe is generated upon the difference between the two successive imageframes, wherein the image processing unit false colors the infrared-onlyimage frame, wherein the image processing unit adds the false coloredinfrared-only image frame back to the image frame generated when thelaser is off, whereby a composite image frame of visible light andinfrared light is generated; and (1) operating an image displaying unitto display images based on the image frames generated from the imageprocessing unit, wherein the image displaying unit is connected to theimage processing unit.

In various embodiments, the present invention provides a non-transitorycomputer-readable storage medium storing one or more programs forimaging a sample comprising an infrared or near-infrared fluorophore,the one or more programs for execution by one or more processors of acomputer system, the one or more programs comprising instructions for:(a) operating an image sensor to detect visible light and infrared lightand generate sensor signals at a first frequency, wherein there is noinfrared filter in the light path from the sample to the image sensor,and wherein the image sensor comprises blue, green and red pixelsensors; (b) operating a laser to emit an excitation light for theinfrared or near-infrared fluorophore and to alternate between on andoff statuses at a second frequency, wherein the second frequency is halfof the first frequency; (c) operating a first channel to conduct theexcitation light from the laser to the sample; (d) operating a lightsource to emit a light comprising visible light; (e) operating a secondchannel to conduct the visible light from the light source to thesample; (f) operating a notch beam splitter in the light path from thelaser to the sample and in the light path from the sample to the imagesensor, whereby the excitation light is reflected by the notch beamsplitter to the sample, whereby the excitation light excites theinfrared or near-infrared fluorophore in the sample to emit an emissionlight, and whereby the emission light is transmitted through the notchbeam splitter to the image sensor; (g) operating a third channel toconduct the emission light from the sample to the image sensor; (h)operating a fourth channel to conduct the visible light from the sampleto the image sensor; (i) operating a notch filter in the light path fromthe sample to the image sensor, whereby the notch filter blocks theexcitation light; (j) operating a synchronization module to synchronizethe image sensor with the laser and visible light, whereby a singlesensor signal is synchronized to a single on or off status of the laser;(k) operating an image processing unit to process sensor signals togenerate image frames, wherein the image processing unit is connected tothe image sensor, wherein the image processing unit subtracts an imageframe generated when the laser is off from the previous or next imageframe generated when the laser is on, whereby an infrared-only imageframe is generated upon the difference between the two successive imageframes, wherein the image processing unit false colors the infrared-onlyimage frame, wherein the image processing unit adds the false coloredinfrared-only image frame back to the image frame generated when thelaser is off, whereby a composite image frame of visible light andinfrared light is generated; and (1) operating an image displaying unitto display images based on the image frames generated from the imageprocessing unit, wherein the image displaying unit is connected to theimage processing unit.

In various embodiments, the present invention provides a computerimplemented method for imaging a sample, comprising: on a device havingone or more processors and a memory storing one or more programs forexecution by the one or more processors, the one or more programsincluding instructions for: providing a sample; providing an imagingsystem of any previous claim; and imaging the sample using the imagingsystem.

In various embodiments, the present invention provides a computer systemfor imaging a sample, comprising: one or more processors and memory tostore one or more programs, the one or more programs comprisinginstructions for: providing a sample; providing an imaging system of anyprevious claim; and imaging the sample using the imaging system.

In various embodiments, the present invention provides a non-transitorycomputer-readable storage medium storing one or more programs forimaging a sample, the one or more programs for execution by one or moreprocessors of a computer system, the one or more programs comprisinginstructions for: providing a sample; providing an imaging system of anyprevious claim; and imaging the sample using the imaging system.

In various embodiments, the present invention provides a computerimplemented method for treating a subject with a tumor, comprising: on adevice having one or more processors and a memory storing one or moreprograms for execution by the one or more processors, the one or moreprograms including instructions for: administering an infrared dye tothe subject, thereby labeling the tumor with the infrared dye;performing a surgery on the subject to access the area of the labeledtumor; providing an imaging system of any previous claim; identifyingthe labeled tumor under the imaging system; and removing the labeledtumor, thereby treating the subject with the tumor.

In various embodiments, the present invention provides a computer systemfor treating a subject with a tumor, comprising: one or more processorsand memory to store one or more programs, the one or more programscomprising instructions for: administering an infrared dye to thesubject, thereby labeling the tumor with the infrared dye; performing asurgery on the subject to access the area of the labeled tumor;providing an imaging system of any previous claim; identifying thelabeled tumor under the imaging system; and removing the labeled tumor,thereby treating the subject with the tumor.

In various embodiments, the present invention provides a non-transitorycomputer-readable storage medium storing one or more programs fortreating a subject with a tumor, the one or more programs for executionby one or more processors of a computer system, the one or more programscomprising instructions for: administering an infrared dye to thesubject, thereby labeling the tumor with the infrared dye; performing asurgery on the subject to access the area of the labeled tumor;providing an imaging system of any previous claim; identifying thelabeled tumor under the imaging system; and removing the labeled tumor,thereby treating the subject with the tumor.

In various embodiments, the present invention provides a computerimplemented method for capturing and processing images and for smoothimage display, comprising: on a device having one or more processors anda memory storing one or more programs for execution by the one or moreprocessors, the one or more programs including instructions for:utilizing parallel process software coding; transferring a raw image;and de-mosaicing the raw image to the one or more processors.

The one or more processors can comprise a graphics processing unit(GPU).

The parallel process software coding can comprise GPU based ComputerUnified Device Architecture (CUDA).

The parallel process software coding can be stored directly on a videocard.

The raw image can be an 8 bit raw image

The images can comprise full high definition frames at 300 frames persecond, a full HD (1080p) 8 bit image can be approximately 2 Mb in size,the PCIe 3.0 data transfer rate can be approximately 7 Gb/s, and theimage can be transferred to the GPU in 300 μsec.

After transferring the image to the GPU, an image processing operationcan be performed. The image processing operation can be one or more fromthe group consisting of: Bayer demosaicing, subtracting a scatteredlight image from a fluorescence image, adding Red, Green and Bluechannels of a fluorescence frame, imparting false coloring to afluorescence image, and adding a white light image with a false coloredfluorescence image.

In order to improve speed, instead of returning the image to a systemmemory for display, openGL/directx functions of the GPU can be used todisplay a final image.

Images can be displayed on a medical grade HD quality video monitor.

In various embodiments, the present invention provides a computer systemfor capturing and processing images and for smooth image display,comprising: one or more processors and memory to store one or moreprograms, the one or more programs comprising instructions for:utilizing parallel process software coding; transferring a raw image;and de-mosaicing the raw image to the one or more processors.

The one or more processors can comprise a graphics processing unit(GPU).

The parallel process software coding can comprise GPU based ComputerUnified Device Architecture (CUDA).

The parallel process software coding can be stored directly on a videocard.

The raw image can be an 8 bit raw image

The images can comprise full high definition frames at 300 frames persecond, a full HD (1080p) 8 bit image can be approximately 2 Mb in size,the PCIe 3.0 data transfer rate can be approximately 7 Gb/s, and theimage can be transferred to the GPU in 300 μsec.

After transferring the image to the GPU, an image processing operationcan be performed. The image processing operation can be one or more fromthe group consisting of: Bayer demosaicing, subtracting a scatteredlight image from a fluorescence image, adding Red, Green and Bluechannels of a fluorescence frame, imparting false coloring to afluorescence image, and adding a white light image with a false coloredfluorescence image.

In order to improve speed, instead of returning the image to a systemmemory for display, openGL/directx functions of the GPU can be used todisplay a final image.

Images can be displayed on a medical grade HD quality video monitor.

In various embodiments, the present invention provides a non-transitorycomputer-readable storage medium storing one or more programs forcapturing and processing images and for smooth image display, the one ormore programs for execution by one or more processors of a storagemedium, the one or more programs comprising instructions for: utilizingparallel process software coding; transferring a raw image; andde-mosaicing the raw image to the one or more processors.

The one or more processors can comprise a graphics processing unit(GPU).

The parallel process software coding can comprise GPU based ComputerUnified Device Architecture (CUDA).

The parallel process software coding can be stored directly on a videocard.

The raw image can be an 8 bit raw image

The images can comprise full high definition frames at 300 frames persecond, a full HD (1080p) 8 bit image can be approximately 2 Mb in size,the PCIe 3.0 data transfer rate can be approximately 7 Gb/s, and theimage can be transferred to the GPU in 300 μsec.

After transferring the image to the GPU, an image processing operationcan be performed. The image processing operation can be one or more fromthe group consisting of: Bayer demosaicing, subtracting a scatteredlight image from a fluorescence image, adding Red, Green and Bluechannels of a fluorescence frame, imparting false coloring to afluorescence image, and adding a white light image with a false coloredfluorescence image.

In order to improve speed, instead of returning the image to a systemmemory for display, openGL/directx functions of the GPU can be used todisplay a final image.

Images can be displayed on a medical grade HD quality video monitor.

In various embodiments, the present invention provides an imaging systemfor imaging a sample. In accordance with the invention, the samplecomprises an infrared or near-infrared fluorophore. The systemcomprises: an image sensor, a laser, a notch beam splitter, a notchfilter, and a synchronization module. The image sensor detects visiblelight and infrared light and generates sensor signals. The laser emitsan excitation light for the infrared or near-infrared fluorophore andalternates between on and off statuses. The notch beam splitter isplaced in the light path from the laser to the sample and in the lightpath from the sample to the image sensor. The excitation light isreflected by the notch beam splitter to the sample; the excitation lightexcites the infrared or near-infrared fluorophore in the sample to emitan emission light; and the emission light is transmitted through thenotch beam splitter to the image sensor. The notch filter is placed inthe light path from the sample to the image sensor, and the notch filterblocks the excitation light. The synchronization module synchronizes theimage sensor with the laser and visible light, whereby a single sensorsignal is synchronized to a single on or off status of the laser. Invarious embodiments, the imaging system further comprises a fast triggerunit.

In various embodiments, the imaging system further comprises a lightsource to emit a light comprising visible light. In accordance with theinvention, visible light can have a spectrum of 400-700 nm. In someembodiments, there is an infrared filter in the light path from thelight source to the sample. In accordance with the invention, theintensity of the laser is controlled to ensure uniform excitation on thesame area illuminated by visible light.

In accordance with the invention, the on-off frequency of the laser ishalf of the frequency of the image sensor generating sensor signals. Invarious embodiments, the laser alternates between on and off status at afrequency of 60 Hz. In various embodiments, the image sensor generatessensor signals at a frequency of 120 Hz.

In various embodiments, the excitation light comprises light having awavelength of about 785 nm and/or 780 nm. In various embodiments, thenotch beam splitter selectively reflects light having a wavelength ofabout 785 nm and/or 780 nm. In various embodiments, the notch filterblocks light having a wavelength of about 785 nm and/or 780 nm.

In various embodiments, there is no infrared filter in the light pathfrom the sample to the image sensor. In various embodiments, there is noinfrared filter in the light path from the laser to the sample. In someembodiments, there is an optical filter to block the excitation light inthe light path from the sample to the image sensor. In otherembodiments, there is no optical filter to block the excitation light inthe light path from the laser to the sample.

In various embodiments, the imaging system further comprises an imageprocessing unit to process sensor signals to generate image frames. Inaccordance with the present invention, the image processing unit isconnected to the image sensor. In various embodiments, the imageprocessing unit subtracts an image frame generated when the laser is offfrom the previous or next image frame generated when the laser is on,whereby an infrared-only image frame is generated upon the differencebetween the two successive image frames. In accordance with theinvention, the image processing unit false colors the infrared-onlyimage frame. In accordance with the invention, the image processing unitadds the false colored infrared-only image frame back to the image framegenerated when the laser is off, whereby a composite image frame ofvisible light and infrared light is generated. In some embodiments, theimage processing unit generates composite image frames of visible lightand infrared light at a frequency of 60 Hz.

In various embodiments, the imaging system further comprises an imagedisplaying unit to display images based on the image frames generatedfrom the image processing unit. In accordance with the presentinvention, the image displaying unit is connected to the imageprocessing unit. Examples of the image displaying unit include but arenot limited to monitors, projectors, phones, tablets, and screens. Insome embodiments, the image displaying unit displays composite imageframes of visible light and infrared light at a frequency of 60 Hz.

In various embodiments, the imaging system further comprises a firstchannel to conduct the excitation light from the laser to the sample, asecond channel to conduct the visible light from the light source to thesample, a third channel to conduct the emission light from the sample tothe image sensor, and a fourth channel to conduct the visible light fromthe sample to the image sensor. In accordance with the presentinvention, the first, second, third and fourth channels are fourseparate channels or combined into one, two, or three channels. Still inaccordance with the present invention, two or more of the four channelsmay overlap partially or completely on their light paths. In variousembodiments, the first, second, third and fourth channels are endoscopeor microscope.

In various embodiments, the present invention provides an imaging systemfor imaging a sample. In accordance with the invention, the samplecomprises an infrared or near-infrared fluorophore. Still in accordancewith the invention, the infrared or near-infrared fluorophore can be acyanide-based infrared or near-infrared fluorophore (e.g., indocyaninegreen (ICG)). The system comprises: (a) an image sensor, (b) a laser,(c) a first channel, (d) a light source, (e) a second channel, (f) anotch beam splitter, (g) a third channel, (h) a fourth channel, (i) anotch filter, (j) a synchronization module, (k) an image processingunit, and (1) an image displaying unit. (a) The image sensor detectsvisible light and infrared light and generates sensor signals at a firstfrequency. There is no infrared filter in the light path from the sampleto the image sensor. The image sensor comprises blue, green and redpixel sensors. Examples of the image sensor include but are not limitedto CCD image sensors and CMOS image sensors. (b) The laser emits anexcitation light for the infrared or near-infrared fluorophore andalternates between on and off statuses at a second frequency, whereinthe second frequency is half of the first frequency. (c) The firstchannel conducts the excitation light from the laser to the sample. (d)The light source emits a light comprising visible light. (e) The secondchannel conducts the visible light from the light source to the sample.(f) The notch beam splitter is placed in the light path from the laserto the sample and in the light path from the sample to the image sensor.The excitation light is reflected by the notch beam splitter to thesample; the excitation light excites the infrared or near-infraredfluorophore in the sample to emit an emission light; and the emissionlight is transmitted through the notch beam splitter to the imagesensor. (g) The third channel conducts the emission light from thesample to the image sensor. (h) The fourth channel conducts the visiblelight from the sample to the image sensor. (i) The notch filter isplaced in the light path from the sample to the image sensor, and thenotch filter blocks the excitation light. (j) The synchronization modulesynchronizes the image sensor with the laser and visible light, wherebya single sensor signal is synchronized to a single on or off status ofthe laser. (k) The image processing unit is connected to the imagesensor and processes sensor signals to generate image frames. The imageprocessing unit subtracts an image frame generated when the laser is offfrom the previous or next image frame generated when the laser is on,whereby an infrared-only image frame is generated upon the differencebetween the two successive image frames. The image processing unit falsecolors the infrared-only image frame. The image processing unit adds thefalse colored infrared-only image frame back to the image framegenerated when the laser is off, whereby a composite image frame ofvisible light and infrared light is generated. (1) The image displayingunit is connected to the image processing unit and displays images basedon the image frames generated from the image processing unit.

In various embodiments, the image sensor comprises blue, green and redpixel sensors. In one embodiment, all the blue, green and red pixelsensors are sensitive to both visible light and infrared light. Invarious embodiments, the image sensor is a CCD image sensor that detectsvisible light and infrared light and generates CCD image signals. Invarious embodiments, the image sensor is a CMOS image sensor thatdetects visible light and infrared light and generates CMOS imagesignals. In various embodiments, the image sensor is without a NIR longpass filter.

In various embodiments, the present invention provides a method ofimaging a sample. The method comprises the steps of: providing a sample,providing an imaging system described herein, and imaging the sampleusing the imaging system. In further embodiments, the method furthercomprises a step of performing a surgery on a subject to access thesample or to isolate the sample. In various embodiments, the subject hascancer and may need surgery to remove cancerous tissue, and the samplerefers to the body part containing cancerous tissue. In variousembodiments, the subject is a human. In various embodiments, the subjectis a mammalian subject including but not limited to human, monkey, ape,dog, cat, cow, horse, goat, pig, rabbit, mouse and rat. Still in furtherembodiments, the method further comprises a step of labeling the samplewith an infrared or near-infrared fluorophore. In accordance with theinvention, the infrared or near-infrared fluorophore can be indocyaninegreen (ICG), or any suitable cyanide-based infrared or near-infraredfluorophore. In some embodiments, the various infrared or near-infraredfluorophores described herein may be modified to be more or lesslipophilic.

In various embodiments, the present invention also provides a method oftreating a subject with a tumor. The method comprises the steps of:administering an infrared dye to the subject, thereby labeling the tumorwith the infrared dye; performing a surgery on the subject to access thearea of the labeled tumor; providing an imaging system described herein;identifying the labeled tumor under the imaging system; and removing thelabeled tumor, thereby treating the subject with the tumor.

The imaging systems and methods of the invention can be used to image asample from various subjects including but not limited to humans andnonhuman primates such as chimpanzees and other ape and monkey species;farm animals such as cattle, sheep, pigs, goats and horses; domesticmammals such as dogs and cats; laboratory animals including rodents suchas mice, rats and guinea pigs, and the like. In various embodiments, thesubject has cancer and may need surgery to remove cancerous tissue, andthe sample refers to the body part containing cancerous tissue. Invarious embodiments, the sample is a tumor, cell, tissue, organ, or bodypart. In some embodiments, the sample is isolated from a subject. Inother embodiments, the sample is integral of a subject. In accordancewith the invention, the sample comprises an infrared or near-infraredfluorophore.

Examples of the infrared or near-infrared fluorophore include but arenot limited to a cyanide-based infrared or near-infrared fluorophore,indocyanine green (ICG), IR800, Alexa680, and cy5.5, and theirfunctional equivalents, analogs, derivatives or salts. In someembodiments, the various infrared or near-infrared fluorophoresdescribed herein may be modified to be more or less lipophilic. One ofordinary skill in the art would know how to choose suitable elements inthe imaging methods and systems described herein for a particularinfrared or near-infrared fluorophore. As one non-limiting example, whenthe infrared dye to be detected is ICG (excitation 748-789 nm with apeak at 785 nm; emission 814-851 nm with a peak at 825 nm), one ofordinary skill in the art would choose a laser emitting an excitationlight of about 785 nm, a laser clean-up filter transmitting light of775-795 nm, a notch filter blocking light of 770-800 nm, and/or a notchbeam splitter splitting light at 700 nm in various systems and methodsdescribed herein. It is known that ICG has different peaks in differentmaterials. Also, ICG is a non-limiting example and other fluorophoresmay be used in place of ICG. One of ordinary skill in the art wouldunderstand the settings may be modified accordingly when the peak is not785 as described in this non-limiting example. For instance, the systemmay use almost any IR or NIR wavelength by changing the laser excitationand the optical filters.

Typical dosages of an effective amount of the infrared or near-infraredfluorophore can be in the ranges recommended by the manufacturer whereknown imaging compounds are used, and also as indicated to the skilledartisan by the in vitro results in cells or in vivo results in animalmodels. Such dosages typically can be reduced by up to about an order ofmagnitude in concentration or amount without losing relevant labelingactivity. The actual dosage can depend upon the judgment of thephysician, the condition of the patient, and the effectiveness of theimaging method based, for example, on the in vitro results of relevantcultured cells or histocultured tissue sample, or the in vivo resultsobserved in the appropriate animal models. In various embodiments, theinfrared or near-infrared fluorophore may be administered once a day(SID/QD), twice a day (BID), three times a day (TID), four times a day(QID), or more, so as to administer an effective amount of the infraredor near-infrared fluorophore to the subject, where the effective amountis any one or more of the doses described herein.

In various embodiments, the infrared or near-infrared fluorophore isadministered to a subject or applied to a sample about 5-10, 10-20,20-30, or 30-60 minutes before imaging. In various embodiments, theinfrared or near-infrared fluorophore is administered to a subject orapplied to a sample about 1-6, 6-12, 12-18, 18-24, 24-30, 30-36, 36-42,or 42-48 hours before imaging. In an embodiment, the infrared ornear-infrared fluorophore is ICG, or a functional equivalent, analog,derivative or salt of ICG. In other embodiments, the infrared ornear-infrared fluorophore is one from the group consisting of: IR800,Alexa680, cy5.5, a functional equivalent of IR800, a functionalequivalent of Alexa680, a functional equivalent of cy5.5, an analog ofIR800, an analog of Alexa680, an analog of cy5.5, a derivative of IR800,a derivative of Alexa680, a derivative of cy5.5, a salt of IR800, a saltof Alexa 680 or a salt of cy5.5. In certain embodiments, the infrared ornear-infrared fluorophore is administered to a human.

In various embodiments, the infrared or near-infrared fluorophore isadministered to a subject or applied to a sample at about 0.1-0.5,0.5-1, 1-1.5, 1.5-2, 2-3, 3-4, 4-5, 5-10, 10-20, 20-50, or 50-100 mg/kg.In various embodiments, the infrared or near-infrared fluorophore isadministered to a subject or applied to a sample at about 0.001 to 0.01mg/kg, 0.01 to 0.1 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 5 mg/kg, 5 to 10mg/kg, 10 to 20 mg/kg, 20 to 50 mg/kg, 50 to 100 mg/kg, 100 to 200mg/kg, 200 to 300 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 600mg/kg, 600 to 700 mg/kg, 700 to 800 mg/kg, 800 to 900 mg/kg, or 900 to1000 mg/kg. Here, “mg/kg” refers to mg per kg body weight of thesubject. In an embodiment, the infrared or near-infrared fluorophore isICG, or a functional equivalent, analog, derivative or salt of ICG. Inother embodiments, the infrared or near-infrared fluorophore is one fromthe group consisting of: IR800, Alexa680, cy5.5, a functional equivalentof IR800, a functional equivalent of Alexa680, a functional equivalentof cy5.5, an analog of IR800, an analog of Alexa680, an analog of cy5.5,a derivative of IR800, a derivative of Alexa680, a derivative of cy5.5,a salt of IR800, a salt of Alexa 680 or a salt of cy5.5. In certainembodiments, the infrared or near-infrared fluorophore is administeredto a human.

In various embodiments, the infrared or near-infrared fluorophore isadministered to a subject or applied to a sample once, twice, three ormore times. In various embodiments, the infrared or near-infraredfluorophore is administered to a subject or applied to a sample about1-3 times per day, 1-7 times per week, or 1-9 times per month. Still insome embodiments, the infrared or near-infrared fluorophore isadministered to a subject or applied to a sample for about 1-10 days,10-20 days, 20-30 days, 30-40 days, 40-50 days, 50-60 days, 60-70 days,70-80 days, 80-90 days, 90-100 days, 1-6 months, 6-12 months, or 1-5years. In an embodiment, the infrared or near-infrared fluorophore isICG, or a functional equivalent, analog, derivative or salt of ICG. Incertain embodiments, the infrared or near-infrared fluorophore isadministered to a human.

In accordance with the invention, the infrared or near-infraredfluorophore may be administered using the appropriate modes ofadministration, for instance, the modes of administration recommended bythe manufacturer. In accordance with the invention, various routes maybe utilized to administer the infrared or near-infrared fluorophore ofthe claimed methods, including but not limited to aerosol, nasal, oral,transmucosal, transdermal, parenteral, implantable pump, continuousinfusion, topical application, capsules and/or injections. In variousembodiments, the retinoid agonist is administered intravascularly,intravenously, intraarterially, intratumorally, intramuscularly,subcutaneously, intranasally, intraperitoneally, or orally.

In various embodiments, the infrared or near-infrared fluorophore isprovides as a pharmaceutical composition. Preferred compositions willalso exhibit minimal toxicity when administered to a mammal.

In various embodiments, the pharmaceutical compositions according to theinvention may be formulated for delivery via any route ofadministration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited toaerosol, nasal, oral, transmucosal, transdermal, parenteral, enteral,topical or local. “Parenteral” refers to a route of administration thatis generally associated with injection, including intraorbital,infusion, intraarterial, intracapsular, intracardiac, intradermal,intramuscular, intraperitoneal, intrapulmonary, intraspinal,intrasternal, intrathecal, intrauterine, intravenous, subarachnoid,subcapsular, subcutaneous, transmucosal, or transtracheal. Via theparenteral route, the compositions may be in the form of solutions orsuspensions for infusion or for injection, or as lyophilized powders.Via the parenteral route, the compositions may be in the form ofsolutions or suspensions for infusion or for injection. Via the enteralroute, the pharmaceutical compositions can be in the form of tablets,gel capsules, sugar-coated tablets, syrups, suspensions, solutions,powders, granules, emulsions, microspheres or nanospheres or lipidvesicles or polymer vesicles allowing controlled release. Typically, thecompositions are administered by injection. Methods for theseadministrations are known to one skilled in the art. In accordance withthe invention, the pharmaceutical composition may be formulated forintravenous, intramuscular, subcutaneous, intraperitoneal, oral or viainhalation administration.

In various embodiments, the pharmaceutical compositions according to theinvention can contain any pharmaceutically acceptable excipient.“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients may be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous. Examples of excipients include but are notlimited to starches, sugars, microcrystalline cellulose, diluents,granulating agents, lubricants, binders, disintegrating agents, wettingagents, emulsifiers, coloring agents, release agents, coating agents,sweetening agents, flavoring agents, perfuming agents, preservatives,antioxidants, plasticizers, gelling agents, thickeners, hardeners,setting agents, suspending agents, surfactants, humectants, carriers,stabilizers, and combinations thereof.

In various embodiments, the pharmaceutical compositions according to theinvention can contain any pharmaceutically acceptable carrier.“Pharmaceutically acceptable carrier” as used herein refers to apharmaceutically acceptable material, composition, or vehicle that isinvolved in carrying or transporting a compound of interest from onetissue, organ, or portion of the body to another tissue, organ, orportion of the body. For example, the carrier may be a liquid or solidfiller, diluent, excipient, solvent, or encapsulating material, or acombination thereof. Each component of the carrier must be“pharmaceutically acceptable” in that it must be compatible with theother ingredients of the formulation. It must also be suitable for usein contact with any tissues or organs with which it may come in contact,meaning that it must not carry a risk of toxicity, irritation, allergicresponse, immunogenicity, or any other complication that excessivelyoutweighs its therapeutic benefits.

The pharmaceutical compositions according to the invention can also beencapsulated, tableted or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers maybe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Liquid carriers include syrup, peanutoil, olive oil, glycerin, saline, alcohols and water. Solid carriersinclude starch, lactose, calcium sulfate, dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. The carrier may also include a sustained release material suchas glyceryl monostearate or glyceryl distearate, alone or with a wax.

The pharmaceutical preparations are made following the conventionaltechniques of pharmacy involving milling, mixing, granulation, andcompressing, when necessary, for tablet forms; or milling, mixing andfilling for hard gelatin capsule forms. When a liquid carrier is used,the preparation will be in the form of a syrup, elixir, emulsion or anaqueous or non-aqueous suspension. Such a liquid formulation may beadministered directly p.o. or filled into a soft gelatin capsule.

The pharmaceutical compositions according to the invention may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of labeling asample in a given subject. This amount will vary depending upon avariety of factors, including but not limited to the characteristics ofthe labeling compound such as an infrared or near-infrared fluorophore,(including activity, pharmacokinetics, pharmacodynamics, andbioavailability), the physiological condition of the subject (includingage, sex, disease type and stage, general physical condition,responsiveness to a given dosage, and type of medication), the nature ofthe pharmaceutically acceptable carrier or carriers in the formulation,and the route of administration. One skilled in the clinical andpharmacological arts will be able to determine a effective amount forlabeling a sample through routine experimentation, for instance, bymonitoring a subject's response to administration of a compound andadjusting the dosage accordingly. For additional guidance, seeRemington: The Science and Practice of Pharmacy (Gennaro ed. 20thedition, Williams & Wilkins PA, USA) (2000).

Before administration to a subject, formulants may be added to thecomposition. A liquid formulation may be preferred. For example, theseformulants may include oils, polymers, vitamins, carbohydrates, aminoacids, salts, buffers, albumin, surfactants, bulking agents orcombinations thereof.

Carbohydrate formulants include sugar or sugar alcohols such asmonosaccharides, disaccharides, or polysaccharides, or water solubleglucans. The saccharides or glucans can include fructose, dextrose,lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran,pullulan, dextrin, alpha and beta cyclodextrin, soluble starch,hydroxethyl starch and carboxymethylcellulose, or mixtures thereof.“Sugar alcohol” is defined as a C4 to C8 hydrocarbon having an —OH groupand includes galactitol, inositol, mannitol, xylitol, sorbitol,glycerol, and arabitol. These sugars or sugar alcohols mentioned abovemay be used individually or in combination. There is no fixed limit toamount used as long as the sugar or sugar alcohol is soluble in theaqueous preparation. In one embodiment, the sugar or sugar alcoholconcentration is between 1.0 w/v % and 7.0 w/v %, more preferablebetween 2.0 and 6.0 w/v %. Amino acids formulants include levorotary (L)forms of carnitine, arginine, and betaine; however, other amino acidsmay be added. In some embodiments, polymers as formulants includepolyvinylpyrrolidone (PVP) with an average molecular weight between2,000 and 3,000, or polyethylene glycol (PEG) with an average molecularweight between 3,000 and 5,000.

It is also preferred to use a buffer in the composition to minimize pHchanges in the solution before lyophilization or after reconstitution.Most any physiological buffer may be used including but not limited tocitrate, phosphate, succinate, and glutamate buffers or mixturesthereof. In some embodiments, the concentration is from 0.01 to 0.3molar. Surfactants that can be added to the formulation are shown in EPNos. 270,799 and 268,110.

Another drug delivery system for increasing circulatory half-life is theliposome. Methods of preparing liposome delivery systems are discussedin Gabizon et al., Cancer Research (1982) 42:4734; Cafiso, BiochemBiophys Acta (1981) 649:129; and Szoka, Ann Rev Biophys Eng (1980)9:467. Other drug delivery systems are known in the art and aredescribed in, e.g., Poznansky et al., DRUG DELIVERY SYSTEMS (R. L.Juliano, ed., Oxford, N. Y. 1980), pp. 253-315; M. L. Poznansky, PharmRevs (1984) 36:277.

After the liquid pharmaceutical composition is prepared, it may belyophilized to prevent degradation and to preserve sterility. Methodsfor lyophilizing liquid compositions are known to those of ordinaryskill in the art. Just prior to use, the composition may bereconstituted with a sterile diluent (Ringer's solution, distilledwater, or sterile saline, for example) which may include additionalingredients. Upon reconstitution, the composition is administered tosubjects using those methods that are known to those skilled in the art.

The compositions of the invention may be sterilized by conventional,well-known sterilization techniques. The resulting solutions may bepackaged for use or filtered under aseptic conditions and lyophilized,the lyophilized preparation being combined with a sterile solution priorto administration. The compositions may containpharmaceutically-acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, and stabilizers (e.g., 1-20% maltose, etc.).

In some embodiments, the invention described herein is provided with acustom lens solution (e.g., a camera), for example, as a complete systemcontaining all components for usage. In other embodiments, the inventiondescribed herein is provided to complement a user's existing equipment,for example, as an add-on system to be used with NIR-capable exoscopesand endoscopes, or to be integrated into operating microscopes.

While various embodiments of the present invention are described in thecontext of various infrared or near-infrared fluorophores, it should notbe construed that the present invention is limited to those infrared ornear-infrared fluorophores. In fact, those infrared or near-infraredfluorophores are merely non-limiting examples. Indeed, the presentinvention may be used for fluorophores in any suitable segment ofelectromagnetic spectrum, for example, ultraviolet (UV), ultraviolet A,ultraviolet B, ultraviolet C, near ultraviolet, middle ultraviolet, farultraviolet, hydrogen lyman-alpha, vacuum ultraviolet, extremeultraviolet, visible, infrared, near infrared, mid infrared, and farinfrared. Examples of fluorophores outside the infrared or near-infraredrange include but are not limited to fluorescein, sodium yellow, and5-aminolevulinic acid (5-ALA). While in various embodiments of thepresent invention, particular types of imaging components (e.g., imagesensors, lasers, laser clean-up filters, notch filters, and otherassociated filters) are described in the context of various infrared ornear-infrared fluorophores, it should not be construed that the presentinvention is limited to those particular imaging components. In fact,those particular imaging components are merely non-limiting examples.Indeed, the present invention also contemplates choosing and includingappropriate imaging components (e.g., image sensors, lasers, laserclean-up filters, notch filters, and other associated filters) for theuse of those fluorophores outside the infrared or near-infrared range.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1

Charged Coupled Devices (CCDs) or Complementarymetal-oxide-semiconductor (CMOS) sensors used in the cameras have abroad spectrum of sensitivity ranging from 400 nm to 1000 nm (FIG. 2).All the Red, Green and Blue sensors show sensitivity in the 800-1000 nmof wavelength. The commercially available cameras have a color filterarray (CFA) or color filter mosaic (CFM) as shown in FIG. 3 on top of asensor to collect color information from the image. In addition to thisfilter array there is an additional NIR short pass filter to cutofflight from 700-1000 nm of wavelength.

Example 2

We use the sensitivity of Red, Green and Blue pixels in near infraredregion (NIR) to detect infrared fluorescence. A visible light sourceilluminates the sample of interest. Also, a laser is used as theexcitation light for the infrared fluorophore in tissue, and theemission light from the infrared fluorophore is detected by a CCDcamera. Meanwhile, the excitation light is filtered before reaching theCCD camera to avoid interfering detection of the emission light. Animage frame is captured when the laser is on (on-frame). Another imageframe is captured when the laser is off (off-frame). The on-framedetects both visible light and infrared fluorescence, while theoff-frame detects only visible light. Thus, the difference in theintensity between the on-frame and off-frame provides information aboutthe infrared fluorescence signal. (FIG. 4).

1. Excitation:

Excitation is achieved using a very narrow wavelength laser @ NIRwavelength (high absorption) 780 or 785 nm. The laser light is passedthrough a special lens where the excitation light it is added per focalusing a notch beam splitter (e.g. NFD01-785-25x36) (FIG. 4). The laseris turned on and off at half the frequency of the camera frame rate. Thelaser intensity can be controlled in order to ensure uniform excitationon the same area visible by the camera.

2. Triggering and Synchronizing:

The laser light is triggered using external trigger which issynchronized with image frames captured by the CCD camera. Every frameof the CCD camera is synchronized with turning on and off of the laser(FIG. 4).

3. CCD:

The frame exposure is controlled using external trigger. As an example,Frame 1 is captured when the laser is off and Frame 2 is captured whenthe laser is on. Frame 1 captures the normal visible light coming fromthe tissue (FIG. 5A). Frame 2 captures additional infrared fluorescence(the white window in FIG. 5B). By subtracting Frame 1 from Frame 2, werecover the additional intensity added by infrared fluorescence. Thiscalculated infrared fluorescence can be given a false color and addedback into Frame 1 to display a composite image frame of visible lightand infrared fluorescence. This process is continuously repeated todisplay or record a real-time video during a surgical operation.

Example 3

By removing the NIR short pass filter in front of the sensor, it ispossible to detect fluorescence light emitted by the NIR fluorophores onall RGB channels (FIG. 2). But in order to differentiate between thevisible light and NIR light we have to ensure that there is no visiblelight on the sensor when capturing an NIR image frame. In order tocapture the NIR light, there should not be any visible light. In somesituations, we capture one frame when there is no visible light or NIRlight, record the light, and then subtract it from the NIR capturedframe. A clinical prototype is shown in FIGS. 6A-6C.

1. Filter Combination:

We use a very specific filter combination to achieve highest signal tonoise ratio (SNR). Instead of using a broadband excitation as describedin most current NIR system, we use an extremely narrow band excitationat 785 nm (optimal for ICG, may vary depending on the fluorophore), theexcitation is further narrowed using a laser clean up filter (FIG. 7)and the excitation light from the fluorescence light coming back fromthe target is removed using a notch filter which is slightly broaderthan the laser clean up filter. This makes sure that we capture theentire fluorescence signal without losing the fluorescence from the areashaded in FIG. 1.

2. Lens System:

The lens system accomplishes two goals: 1) delivery of the pulsed NIRexcitation light and white light to the distal end of the lens to ensurefull illumination of the surgical field and reduce the strength of theexcitation light in the optical pathway of the emission light. Thecasing to this lens system has been designed to deliver both NIR andwhite light to the surgical field in a uniform way. 2) Apochromaticlenses ensure maximal light capture and transmission to the camera, witha built in notch filter (Semrock, 785 nm StopLine® single-notch filter,NF03-785E-25) to remove excitation light.

3. Frame Capture Times:

The frames are captured at very high frame rate of 300 frames per secondusing a frame grabber. Slower or faster frame rate can also be used. Theframe capture and laser strobe (on/off) are synchronized using amultifunction DAQ. This allows us to capture 10 frames for every framefinally displayed (30 fps). The 10 frames are divided in two sets of 5frames each (FIG. 8). The 5 capture frames are further divided as, 1)first frame is WLF (white light “on”, NIR light “off”), 2) the secondframe is a SLF (white light “off”, NIR light “off”), and 3) the nextthree frames are NIF (white light “off”, NIR light “on”). Aftersubtracting SLF from all three NIFs, The NIF RGB channels are addedtogether, and then the final NIF is given false color before adding itto the WLF. Frames generated from both frames are ultimately added toproduce a display frame. This process serves to produce crisp WL and NIRimages at a sufficient video rate to seem instantaneous to the surgeon.The exact order of WLF, SLF and NIF can be shuffled.

4. Computer Architecture, Hardware and Software:

To capture and process full HD frames at 300 frames per second, we mayrely on parallel processing techniques as even the fastest CPUsavailable are unlikely able to perform the required video processingcalculations at a fast enough rate for smooth image display. In order toperform image processing at this frame rate, we can utilize GPU basedComputer Unified Device Architecture (CUDA) parallel process softwarecoding directly on the video card. One of the main limitations of usingCUDA programming is the overheads for the transfer of data from thesystem memory and to the GPU and vice versa. In order to overcome thislimitation our algorithm is designed to transfer a raw 8 bit image priorto de-mosaicing to the GPU. A full HD (1080p) 8 bit image isapproximately 2 Mb in size. If we consider that the PCIe 3.0 datatransfer rate of approximately 7 Gb/s, we can transfer the image to theGPU in 300 μsec. After the image is transferred to the GPU we performimage processing operations such as Bayer demosaicing, subtracting thescattered light image from the fluorescence image, adding the Red, Greenand Blue channels of the fluorescence frame, imparting false coloring tothe fluorescence image, and finally adding the white light image withthe false colored fluorescence image. Lastly, in order to improve thespeed further, instead of returning the image to the system memory fordisplay, we use the openGL/directx functions of the GPU to display thefinal image. Images are displayed on a medical grade HD quality videomonitor. We have already demonstrated the capability to acquire highquality versions of these images and regulate appearance utilizingsoftware.

Example 4

In this non-limiting example shown in FIG. 10, an image sensor ismounted at the end of an endoscope for visualizing tumors endoscopically or laproscopically. The endoscope also have light channels carryingboth white and NIR light. In front of the image sensor, there can be alens or a set of lenses capable of transmitting both visible light andNIR fluorescence light. The NIR excitation light will be blocked by anotch filter in front of the lens or set of lenses. The electricalconnection to the sensor as well as the fiber optics will be housedinside the endoscope.

The various methods and techniques described above provide a number ofways to carry out the application. Of course, it is to be understoodthat not necessarily all objectives or advantages described can beachieved in accordance with any particular embodiment described herein.Thus, for example, those skilled in the art will recognize that themethods can be performed in a manner that achieves or optimizes oneadvantage or group of advantages as taught herein without necessarilyachieving other objectives or advantages as taught or suggested herein.A variety of alternatives are mentioned herein. It is to be understoodthat some preferred embodiments specifically include one, another, orseveral features, while others specifically exclude one, another, orseveral features, while still others mitigate a particular feature byinclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be employed invarious combinations by one of ordinary skill in this art to performmethods in accordance with the principles described herein. Among thevarious elements, features, and steps some will be specifically includedand others specifically excluded in diverse embodiments.

Although the application has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the application extend beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses and modifications and equivalents thereof.

Preferred embodiments of this application are described herein,including the best mode known to the inventors for carrying out theapplication. Variations on those preferred embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. It is contemplated that skilled artisans canemploy such variations as appropriate, and the application can bepracticed otherwise than specifically described herein. Accordingly,many embodiments of this application include all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the application unless otherwise indicated herein orotherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications,and other material, such as articles, books, specifications,publications, documents, things, and/or the like, referenced herein arehereby incorporated herein by this reference in their entirety for allpurposes, excepting any prosecution file history associated with same,any of same that is inconsistent with or in conflict with the presentdocument, or any of same that may have a limiting affect as to thebroadest scope of the claims now or later associated with the presentdocument. By way of example, should there be any inconsistency orconflict between the description, definition, and/or the use of a termassociated with any of the incorporated material and that associatedwith the present document, the description, definition, and/or the useof the term in the present document shall prevail.

It is to be understood that the embodiments of the application disclosedherein are illustrative of the principles of the embodiments of theapplication. Other modifications that can be employed can be within thescope of the application. Thus, by way of example, but not oflimitation, alternative configurations of the embodiments of theapplication can be utilized in accordance with the teachings herein.Accordingly, embodiments of the present application are not limited tothat precisely as shown and described.

Various embodiments of the invention are described above in the DetailedDescription. While these descriptions directly describe the aboveembodiments, it is understood that those skilled in the art may conceivemodifications and/or variations to the specific embodiments shown anddescribed herein. Any such modifications or variations that fall withinthe purview of this description are intended to be included therein aswell. Unless specifically noted, it is the intention of the inventorsthat the words and phrases in the specification and claims be given theordinary and accustomed meanings to those of ordinary skill in theapplicable art(s).

The foregoing description of various embodiments of the invention knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the invention to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe invention and its practical application and to enable others skilledin the art to utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.Therefore, it is intended that the invention not be limited to theparticular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this invention and its broader aspects and,therefore, the appended claims are to encompass within their scope allsuch changes and modifications as are within the true spirit and scopeof this invention.

What is claimed is:
 1. An imaging system for imaging a sample comprisingan infrared or near-infrared fluorophore, comprising: a laser to emit aninfrared (IR) or near-infrared (NIR) excitation light for the infraredor near-infrared fluorophore, wherein the excitation light is conductedto the sample; a laser clean-up filter in the excitation light path fromthe laser to the sample, wherein the laser clean-up filter narrows thewavelength band of the excitation light to the peak absorption band ofthe infrared or near-infrared fluorophore, wherein the narrowedexcitation light excites the infrared or near-infrared fluorophore inthe sample to emit an emission light, wherein the emission light isconducted to an image sensor, and wherein there is no infrared filter inthe emission light path from the sample to the image sensor; a notchfilter in the emission light path from the sample to the image sensor,wherein the notch filter blocks the excitation light; a white lightsource to emit a light comprising visible light, wherein the visiblelight is conducted to the sample, wherein the sample reflects thevisible light, wherein the reflected visible light is conducted to theimage sensor, wherein the image sensor is one image sensor configured todetect both the emission light and the visible light from the sample andconfigured to generate sensor signals, and wherein the image sensorcomprises blue, green and red pixel sensors; and a notch beam splitterin the light path from the laser to the sample and in the light pathfrom the white light source to the sample, whereby the excitation lightis reflected by the notch beam splitter to the sample and the visiblelight is transmitted by the notch beam splitter to the sample.
 2. Theimaging system of claim 1, wherein there is no Fabry-Perot etalon, Ramananalysis filter wheel, dispersive element, dispersive prism, isoscelesprism, diffraction grating, reflection-type diffraction grating, ortransmission-type diffraction grating in the emission light path fromthe sample to the image sensor.
 3. The imaging system of claim 1,wherein the emission light is not dispersed or filtered for Raman bandselection in the emission light path from the sample to the imagesensor.
 4. The imaging system of claim 1, wherein the image sensor isconfigured not to detect Raman scattered light from the sample.
 5. Theimaging system of claim 1, wherein the infrared or near-infraredfluorophore is one from the group consisting of: indocyanine green(ICG), a functional equivalent of ICG, an analog of ICG, a derivative ofICG, a salt of ICG, IR800, Alexa680, cy5.5, a functional equivalent ofIR800, a functional equivalent of Alexa680, a functional equivalent ofcy5.5, an analog of IR800, an analog of Alexa680, an analog of cy5.5, aderivative of IR800, a derivative of Alexa680, a derivative of cy5.5, asalt of IR800, a salt of Alexa 680 or a salt of cy5.5.
 6. The imagingsystem of claim 1, wherein the laser is pulsed.
 7. The imaging system ofclaim 1, wherein the white light source is pulsed.
 8. The imaging systemof claim 1, wherein the image sensor is a CCD image sensor.
 9. Theimaging system of claim 1, wherein the image sensor is a CMOS imagesensor.
 10. The imaging system of claim 1, wherein the laser clean-upfilter is not a spatial filter.
 11. The imaging system of claim 1,wherein the blocking range of the notch filter is broader than thetransmitting range of the laser clean-up filter.
 12. The imaging systemof claim 1, wherein the excitation light comprises light having awavelength of about 785 nm.
 13. The imaging system of claim 1, whereinthe laser clean-up filter selectively transmits light having awavelength of about 785 nm.
 14. The imaging system of claim 1, whereinthe notch filter selectively blocks light having a wavelength of about785 nm.
 15. The imaging system of claim 1, wherein the notch beamsplitter reflects light having a wavelength of about 785 nm.
 16. Theimaging system of claim 1, further comprising an image processing unitto process sensor signals to generate image frames, wherein the imageprocessing unit is connected to the image sensor.
 17. The imaging systemof claim 16, wherein the image processing unit process sensor signals togenerate at least one white light frame (WLF) when the sample receivesonly visible light, at least one stray light frame (SLF) when the samplereceives neither visible light nor the excitation light, and one or morenear infrared frames (NIFs) when the sample receives only excitationlight, and wherein the image processing unit subtracts the SLF from eachNIF and then adds together all SLF-subtracted NIFs to generate a finalNIF.
 18. The imaging system of claim 17, wherein the image processingunit false colors the final NIF.
 19. The imaging system of claim 18,wherein the image processing unit adds the false colored final NIF tothe WLF to generate a composite image frame of visible light andinfrared light.
 20. The imaging system of claim 16, further comprisingan image displaying unit to display images based on the image framesgenerated from the image processing unit, wherein the image displayingunit is connected to the image processing unit.
 21. The imaging systemof claim 1, wherein the excitation light from the laser is conducted tothe sample through a first channel, wherein the visible light from thewhite light source is conducted to the sample through a second channel,wherein the emission light emitted from the sample is conducted to theimage sensor through a third channel, and wherein the visible lightreflected from the sample is conducted to the image sensor through afourth channel.
 22. The imaging system of claim 1, wherein theexcitation light from the laser is conducted to the sample through afirst light channel housed in an endoscope; wherein the visible lightfrom the white light source is conducted to the sample through a secondlight channel housed in the endoscope; and wherein the image sensor ishoused in the endoscope at or near the patient end of the endoscope. 23.The imaging system of claim 22, wherein the first light channel is anoptical cable.
 24. The imaging system of claim 22, wherein the secondlight channel is an optical cable.
 25. The imaging system of claim 22,further comprising one or more lenses in the emission light path and/orthe visible light path from the sample to the image sensor, wherein theone or more lenses are located at or near the patient end of theendoscope.
 26. A method for imaging a sample comprising an infrared ornear-infrared fluorophore, comprising: operating a laser to emit aninfrared (IR) or near-infrared (NIR) excitation light for the infraredor near-infrared fluorophore; operating a notch beam splitter to reflectthe excitation light to the sample; conducting the excitation light tothe sample; operating a laser clean-up filter in the excitation lightpath from the laser to the sample to narrow the wavelength band of theexcitation light to the peak absorption band of the infrared ornear-infrared fluorophore, wherein the narrowed excitation light excitesthe infrared or near-infrared fluorophore in the sample to emit anemission light; conducting the emission light to an image sensor,wherein there is no infrared filter in the emission light path from thesample to the image sensor; operating a notch filter in the emissionlight path from the sample to the image sensor to block the excitationlight; operating a white light source to emit a light comprising visiblelight; operating the notch beam splitter to transmit the visible lightto the sample; conducting the visible light to the sample, wherein thesample reflects the visible light; conducting the reflected visiblelight to the image sensor; and operating the image sensor to detect boththe emission light and the visible light from the sample and to generatesensor signals, wherein the image sensor is one image sensor andcomprises blue, green and red pixel sensors.
 27. The method of claim 26,further comprising performing a surgery on a subject to access thesample or to isolate the sample.
 28. The method of claim 26, furthercomprising labeling the sample with an infrared or near-infraredfluorophore.